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COLORADO DIVISION OF WILDLIFE Prying Into Prions Investigating Chronic Wasting Disease COLORADO DIVISION OF WILDLIFE 6060 Broadway • Denver, CO 80216 (303) 297-1192 • www.wildlife.state.co.us In Dedication to Dr. Elizabeth (“Beth”) Williams Dr. Elizabeth (“Beth”) Williams was a only fitting that the Colorado Division life-long student, contemplating problems of Wildlife dedicates this high school she encountered with an open mind and science education module Prying into viewing them as new opportunities to Prions—Investigating Chronic Wasting learn. Beth’s innate curiosity, combined Disease to the memory of Dr. Beth with her careful and thorough approach Williams. We hope this tool will help to diagnostic pathology, allowed her to stimulate growth and training, and will succeed where others help foster a similar had previously failed in passion for life-long unraveling the cause of a learning in our next mysterious but little-known generation of scientists. disease of captive mule deer called “chronic wasting We also recognize that disease”. The realization that no successful scientist the chronic wasting accomplishes much of Photo by the Rocky Mountain News, provided syndrome seen in captive consequence in isolation, courtesy of Dr. Jean Jewell, University of Wyoming. deer was in fact one of a and thus we want to handful of diseases in the transmissible acknowledge and thank the many other spongiform encephalopathy group researchers, biologists, pathologists, and opened the door to three decades of technicians in Colorado, Wyoming, and research and scientific progress toward a elsewhere who have contributed to the better understanding of chronic wasting body of knowledge on chronic wasting disease, as well as other prion diseases disease and other prion diseases. The of animals and humans. Although notable challenges the Division of Wildlife and progress has been made, there is still other management agencies have faced much work to be done before our in addressing chronic wasting disease knowledge and technology is sufficient to would have been far more daunting in control and manage these diseases with the absence of the firm foundation of effectiveness comparable to our capacity knowledge provided by their collective to deal with diseases caused by more efforts. conventional pathogens. Because future advances likely will depend in no small Michael W. Miller part on the creativity and inquisitiveness Senior Wildlife Veterinarian of a new generation of scientists, it seems Colorado Division of Wildlife Acknowledgments Funding for this project was provided by US Fish & Wildlife Service Wildlife Conservation and Restoration Program Grant No R-11-1, Great Outdoors Colorado Trust Fund (GOCO) and the sportsmen of Colorado. The Colorado Division of Wildlife gratefully acknowledges the following individuals: For content advice and critical review: Dr. Michael W. Miller, DVM Colorado Division of Wildlife, Wildlife Research Center, Fort Collins, CO Dr. Jean E. Jewell, University of Wyoming, Veterinary Science Department, Wyoming State Veterinary Laboratory, Laramie, WY For assistance in developing the field test: Ann Campbell, Legacy High School, Broomfield, CO Scott Campbell, Douglas County High School, Castle Rock, CO Kate Henson, Jefferson Academy, Broomfield, CO Jeff Keidel, Buena Vista High School, Buena Vista, CO Robert Lancaster, Walsh High School, Walsh, CO Nicole Knapp, Science Educator, BSCS Matt Landis, North Park High School, Walden, CO Anne Tweed, Senior Science Consultant, McREL (Mid-continent Research for Education and Learning) Pam Van Scotter, Director, BSCS (Biological Sciences Curriculum Study) Center for Curriculum Development Mark Little, Broomfield High School, Broomfield, CO Rick Moeller, Mountain View High School, Loveland, CO Lyn Neve, Swink High School, Swink, CO Jill Smith, McClave High School, McClave, CO Field-test Educators: Marylin Achten, Aurora Public Schools, Aurora, CO Debbie Yeager, Moffat County High School, Craig, CO Jane Ballard, Berthoud High School, Berthoud, CO Education Advisors and Reviewers: Graphic Design: Project Coordinator and Editor: Lisa Evans, Northeast Region Education Coordinator, DOW Darren Eurich, State of Colorado Integrated Document Solutions (IDS) Wendy Hanophy, DOW Leigh Gillette, Southwest Region Education Coordinator, DOW Linda Groat, Southeast Region Rural Education Specialist, DOW Illustrations: Darren Eurich Renée Herring, Watchable Wildlife Coordinator, DOW Marjorie Leggitt Stan Johnson, Northwest Region Education Coordinator, DOW Classroom Observations and Educator Interviews: Tabbi Kinion, Project WILD Coordinator, DOW Wendy Hanophy, DOW Debbie Lerch-Cushman, Metro Denver Education Coordinator, DOW Steve Lucero, Southeast Region Education Coordinator, DOW Jeff Rucks, Head of Education, DOW James Philips Writers: Wendy Hanophy, DOW Larry Ann Ott, Retired Educator, Adams County School District 12, Colorado Printing: State of Colorado Integrated Document Solutions (IDS) Print Operations Administrative Assistance: Chris DiMarco © Copyright 2007 by Colorado Division of Wildlife. All rights reserved. Permission granted to reproduce handouts for classroom use. All illustrations are copyrighted by the artists and may not be reproduced or transmitted in any form or by any means, electronic or mechanical, or by any information storage or retrieval system, without permission in writing. For permissions and other rights under this copyright, please contact Colorado Division of Wildlife, 6060 Broadway, Denver, CO 80216, Attn: Wendy Hanophy. Table Of Contents Prying Into Prions Investigating Chronic Wasting Disease Module Overview . . . . . . . . . . . . . . 1 Lesson 1: A Brain Wreck Educator’s Overview. . . . . . . . . . . . 6 Molecules And Cells . . . . . . . . . . 10 A Brain Wreck . . . . . . . . . . . . . . . 11 Lesson 2: Pathological Proteins? Educator’s Overview. . . . . . . . . . . 14 Pathological Proteins? . . . . . . . . . 18 Silly, Silly Protein Structure . . . . . 21 Lesson 3: We Moose Crack The Code Educator’s Overview. . . . . . . . . . . 34 We Moose Crack The Code . . . . 87 Decode That Gene . . . . . . . . . . . . 91 Lesson 4: Cannibalism, Forgetfulness, And Sleepless Nights Educator’s Overview. . . . . . . . . . . 94 Cannibalism, Fogetfulness, And Sleepless Nights . . . . . . . . . 98 Table Of Contents Lesson 5: Oh…Deer Educator’s Overview. . . . . . . . . . 104 Oh…Deer . . . . . . . . . . . . . . . . . . 110 Lesson 6: Here To Stay, Not Gone Tomorrow Educator’s Overview. . . . . . . . . . 124 Here To Stay, Not Gone Tomorrow . . . . . . . . . . 130 Glossary . . . . . . . . . . . . . . . . . . . . . . . . . 136 Module Overview Prying Into Prions Investigating Chronic Wasting Disease What is CWD? Chronic wasting disease (CWD) is a fatal neurological disease found in deer, elk and moose. It belongs to a family of diseases known as transmissible spongiform encephalopathies or prion diseases. The disease attacks the brains of infected deer, elk and moose, causing the animals to become emaciated, display abnormal behavior and lose coordination, and eventually die. Chronic Wasting Disease (CWD) has been recognized in free-ranging deer in Colorado since 1985, but how long it has been present or how it arose are unknown. Unlike all infectious diseases known at the time, the pathogen for transmissible spongiform encephalopathies or TSEs was identified as a protein. Stanley Prusiner, the scientist who isolated the protein, named it a “prion,” which is short for “proteinaceaous infectious particle.” The prion protein exists in two forms. The normal form is found in many types of cells, including those in the central nervous system. The pathological prion form has the same chemical composition, but a different shape. This abnormally shaped prion protein damages the brain tissue and converts other normal prion proteins in cells into the same abnormal shape! Why Study CWD? Students are often unenthusiastic about studying the basic chemistry of life—the structure of proteins, lipids, carbohydrates, and nucleic acids. They can be equally apathetic learning about protein synthesis and gene expression. Prion diseases, unfortunately, are fear-provoking and seem to compel human interest, providing a relevant context to study life’s molecules and processes. Why is the Colorado Division of Wildlife Providing this Module? Chronic wasting disease was first noticed by researchers at Colorado’s Foothills Wildlife Research Facility in 1967. Captive mule deer that were being maintained for nutritional studies began to lose weight. They became listless, started slobbering and drooling, and seemed constantly thirsty. Some stopped socializing with other deer. Most of the affected deer died within months. This new affliction was named chronic wasting disease. In 1977, Elizabeth Williams, studying for her doctorate in veterinary medicine at Colorado State University, identified CWD as a new transmissible spongiform encephalopathy. In 1985, CWD was discovered in free-ranging deer and elk in Colorado. That same year, a handful of cattle from various areas in the United Kingdom began dying of a strange illness. Examination of the dead cattle’s brains revealed abnormal, microscopic holes. The new disease was named bovine spongiform encephalopathy or BSE. Prying Into Prions Module Overview The idea that form dictates function is a central tenet of science and biology. There is probably no better group of diseases that illustrate this concept than TSEs such as chronic wasting disease—diseases caused by a simple change in the form of a protein. To understand these diseases, students must first understand what proteins are, how they are made and how they fold and misfold. 1 In 1996, health officials in the United Kingdom discovered a new variant of a human transmissible spongiform encephalopathy called Creutzfeldt-Jakob disease (CJD). CJD normally occurs in older people, but in 1996 a few teenagers started showing classic CJD symptoms. Biopsies of the brain tissue of these victims revealed that the new variant Creutzfeldt-Jakob disease, vCJD, involved the same prion strain as BSE. It became clear that all transmissible spongiform encephalopathies could potentially be a human health problem. The Colorado Division of Wildlife (DOW) has many very good reasons to study CWD and share what is learned with the public. Wildlife biologists want to contain the disease, protect wild herds, and protect the public health. More is currently known about the distribution and ecology of the disease in Colorado than in any other place on earth. The DOW is a leader in investigating the disease, and providing information and education about CWD to the public. In fact, efforts made by the Colorado Division of Wildlife serve as models for other wildlife regulatory agencies. One part of the DOW public information and education effort is to foster an informed citizenry, capable of making good decisions regarding wildlife conservation and management. The DOW hopes that you and your students enjoy learning about CWD and other important wildlife issues, so that you are better able to partner with us in our efforts in Keeping Colorado Wild! Module Overview What is This? 2 This six-lesson module, designed for approximately two weeks of classroom instruction, begins by presenting an interesting group of emerging diseases— transmissible spongiform encephalopathies (TSEs). After students discuss the variety of evidence that scientists must collect to determine the origin, infectious agent, and route of transmission of a transmissible Prying Into Prions disease, they explore the role of proteins in living organisms, the chemistry and behavior of proteins, and the genetic code that creates protein (DNA, transcription, and translation). Throughout the module, they look at and evaluate experimental design. The module is designed to supplement or replace the activities found in most high school biology textbooks that address the chemistry of life, DNA and the genetic code, and protein synthesis. It is the third module developed by the DOW to address the specific learning objectives of high school students. Materials are inquiry based, develop critical thinking skills, supply evidence to support each concept, and include real data from recent research projects. Using Prying into Prions: Investigating Chronic Wasting Disease in Your Classroom The lessons in this module are designed to be taught in sequence. For each activity, students assume the role of researchers studying an emerging disease. As they evaluate and analyze information concerning CWD and other transmissible spongiform encephalopathies, they explore the larger role of proteins in living organisms. There are DOW Education Coordinators throughout the state who may be able to provide additional support or resources on CWD and other biology topics. Feel free to contact your Education Coordinator at any time—they would love to hear from you: Leigh Gillette, Education Coordinator, SW Region 415 Turner Drive Durango, CO 81303 970-375-6709 Stan Johnson, Education Coordinator, NW Region 711 Independent Avenue Grand Junction, CO 81505 970-255-6191 Linda Groat, Rural Education Specialist, SE Region 2500 S. Main Lamar, CO 81502 719-336-6608 Debbie Lerch-Cushman, Education Coordinator, Denver Metro Area 6060 Broadway Denver, CO 80216 303-291-7328 Steve Lucero, Education Coordinator, SE Region 4255 Sinton Road Colorado Springs, CO 80907 719-227-5203 Correlation to the Colorado Model Content Standards Prying into Prions: Investigating Chronic Wasting Disease supports teachers in their efforts to provide the knowledge and skills specified in the Colorado Model Content Standards and the corresponding grade level assessment frameworks. Lisa Evans, Education Coordinator, NE Region 317 W. Prospect Fort Collins, CO 80526 970-472-4343 COLORADO MODEL CONTENT STANDARDS Lesson 1: A Brain Wreck Lesson 2: Pathological Proteins? Lesson 3: We Moose Crack The Code Lesson 4: Cannibalism, Forgetfulness, And Sleepless Nights Lesson 5: Oh…Deer Lesson 6: Here To Stay, Not Gone Tomorrow X X X X X X 2: Students know and understand common properties, forms, and changes in matter and energy. X X 3: Students know and understand the characteristics and structure of living things, the processes of life, and how living things interact with each other and their environment. X X X X X X X X Lesson 4: Cannibalism, Forgetfulness, And Sleepless Nights Lesson 5: Oh…Deer Lesson 6: Here To Stay, Not Gone Tomorrow X X X Science 1: Students apply the processes of scientific investigation and design, conduct, communicate about, and evaluate such investigations. 5: Students understand that the nature of science involves a particular way of building knowledge and making meaning of the natural world. X X COLORADO MODEL CONTENT STANDARDS Lesson 1: A Brain Wreck History 4.1: Students understand the impact of scientific and technological developments on individuals and societies. X Lesson 2: Pathological Proteins? Lesson 3: We Moose Crack The Code Module Overview Prying Into Prions 3 COLORADO MODEL CONTENT STANDARDS Lesson 4: Cannibalism, Forgetfulness, And Sleepless Nights Lesson 5: Oh…Deer 1: Students develop number sense and use numbers and number relationships in problem-solving situations and communicate the reasoning used in solving these problems. X X 6: Students link concepts and procedures as they develop and use computational techniques, including estimation, mental arithmetic, paper-and-pencil, calculators, and computers, in problem-solving situations and communicate the reasoning used in solving these problems. X Lesson 1: A Brain Wreck Mathematics Lesson 2: Pathological Proteins? Lesson 3: We Moose Crack The Code Lesson 6: Here To Stay, Not Gone Tomorrow COLORADO MODEL CONTENT STANDARDS Lesson 1: A Brain Wreck Lesson 2: Pathological Proteins? Lesson 3: We Moose Crack The Code Lesson 4: Cannibalism, Forgetfulness, And Sleepless Nights Lesson 5: Oh…Deer Lesson 6: Here To Stay, Not Gone Tomorrow 1: Students read and understand a variety of materials. X X X X X X 2: Students write and speak for a variety of purposes and audiences. X 3: Students write and speak using conventional grammar, usage, sentence structure, punctuation, capitalization, and spelling. X X X X X X 4: Students apply thinking skills to their reading, writing, speaking, listening, and viewing. X X X X X X 5: Students read to locate, select, and make use of relevant information from a variety of media, reference, and technological sources. X X X X Lesson 5: Oh…Deer Lesson 6: Here To Stay, Not Gone Tomorrow X X X X X X X X Reading and Writing COLORADO MODEL CONTENT STANDARDS Lesson 1: A Brain Wreck Civics 2.1 Students know the organization and functions of local, state, and national governments. Lesson 2: Pathological Proteins? Lesson 3: We Moose Crack The Code Lesson 4: Cannibalism, Forgetfulness, And Sleepless Nights X 2.4 Students know how public policy is developed at the local, state, and national levels. 3.1 Students know how and why governments and nongovernmental agencies around the world interact politically. Module Overview 3.3 Students understand the domestic and foreign policy influence the United States has on other nations and how the actions of other nations influence politics and society of the United States. 4 Prying Into Prions X Notes ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ Prying Into Prions Module Overview ________________________________________ 5 Educator’s Overview Lesson 1 A Brain Wreck Summary Duration One or two 45-minute class periods Students read an article about an interesting group of emerging diseases—transmissible spongiform encephalopathies (TSEs). They reflect upon and discuss what they already know and understand about disease to prepare themselves to study TSEs. Learning Objectives After completing this activity, students will be able to: Vocabulary Bovine spongiform encephalopathy (BSE) Creutzfeldt-Jakob disease (CJD) Emerging disease Infect Pathogen Sporadic disease Transmissible spongiform encephalopathies (TSEs) Variant CreutzfeldtJakob disease (vCJD) Zoonotic • Explain why a state wildlife agency might devote considerable resources—time, money, and people—to study these diseases. • Reflect upon and discuss their prior knowledge about the cause, transmission, and treatment of disease. • Recognize the variety of evidence that scientists must collect to determine the origin, infectious agent, and route of transmission of a transmissible disease. Background Lesson 1: A Brain Wreck 6 • Explain why several diseases have been grouped together in a category called transmissible spongiform encephalopathies (TSEs). This lesson is designed to engage your students’ interest in an unusual group of diseases— transmissible spongiform encephalopathies—and prepare Prying Into Prions them for the exciting activities in this module. The reading and questions are designed to make connections between their past and present learning experiences. Students will apply their knowledge about infectious or transmissible diseases to the scientific research of a new disease. Transmissible spongiform encephalopathies (TSEs) are a rare group of degenerative diseases that affect the brain and central nervous system. All are untreatable and fatal. These diseases are transmissible because they are capable of being transferred from one animal to another, spongiform because they cause the appearance of microscopic sponge-like holes in the brain of the affected animals, and encephalopatic because they are neurodegenerative diseases of the brain. TSEs have been found in many mammals, including humans. The first known TSE, scrapie, was described in sheep in Great Britain and Western Europe over 250 years ago. The name scrapie comes from one of the symptoms of the disease. In addition to excessive lipsmacking, strange walking gaits, and convulsions, sheep with this disorder appear to itch. The animals will compulsively scrape off their fleece against rocks, fences, or trees. Another TSE of livestock was discovered in Great Britain in the early 1980s—bovine spongiform encephalopathy (BSE)— commonly called “mad cow disease.” Nearly 200,000 cattle in Great Britain and thousands in other countries have since succumbed to BSE. Creutzfeldt-Jakob disease (CJD) is the most well-known of the human TSEs. It is a rare type of dementia that affects about one in every one million people worldwide each year. Other human TSEs include kuru, fatal familial insomnia (FFI), and Gerstmann-Straussler-Scheinker disease (GSS). Kuru was identified in people of an isolated tribe in Papua New Guinea and has now almost disappeared. FFI and GSS are extremely rare hereditary diseases, found in just a few families around the world. Another TSE, chronic wasting disease or CWD, was first noted in Colorado in the late 1960s Chronic wasting disease is an important wildlife health issue, and potentially a human health issue. Researchers at the Colorado Division of Wildlife have aggressively studied this disease and have worked to contain it. In addition, the agency has worked hard to keep the public safe and informed. All new developments and understandings about CWD are shared with the public. All hunters can test their animals for CWD and can decide whether to consume or dispose of the meat. This is a new—emerging— disease, and there is still much to learn. Teaching Strategies 1. Thoroughly read the student materials for Molecules And Cells, and A Brain Wreck. 2. Optional: There are some fundamental concepts about the biochemical nature of life that your students must understand before beginning this module. Students’ prior knowledge should include these ideas: Materials and Preparation • Optional: Student Activity Page: Molecules And Cells—one photocopy per student • Student Reading Pages: A Brain Wreck—one photocopy per student • A large, visible writing surface (i.e. chalkboard, whiteboard, butcher paper) • Living things are made up of molecules, which are made of various atoms. • Atoms join together to make molecules by sharing or giving up electrons. Lesson 1: A Brain Wreck A new type of CJD, called variant CJD (vCJD), was first described in 1996 and has been found in Great Britain and several other European countries. The initial symptoms of vCJD are different from those of classic CJD and the disorder typically occurs in younger patients. Research suggested that vCJD resulted from human infection with BSE. in captive deer. It has been widespread in free-ranging deer and elk in northcentral Colorado and southeastern Wyoming since at least the early 1980s. Recently, the disease has also been discovered in moose in Colorado. • The chemical properties of molecules are determined by their ability to bond with other molecules. Prying Into Prions 7 This may seem straightforward, but it isn’t. Students don’t always know what we think they know. Many students, even in high school, have misconceptions about atoms and molecules and their role in living organisms. High school students often have difficulty thinking about living organisms as chemical systems. They often confine the concept of molecules to things encountered in physics and chemistry. Many may think that living organisms are not made up of molecules, only cells. Specific to proteins, students may think that proteins are made up of cells and that molecules of protein are bigger than the size of a cell. Or, they may only associate protein with diet—things that they eat—such as meats, cheeses, etc. If you suspect that your students may have these misconceptions, or are just curious, you may want to have your students do the activity page Molecules And Cells before beginning this module. Discuss the answers provided in the key and clarify any misconceptions. This activity could also be done right before the second lesson in this module. 3. Give each student a copy of the student reading pages: A Brain Wreck. 4. After giving students sufficient time to read or after reading the material as a class, ask students to work in groups of three or four to brainstorm answers to each question. Instruct each student in the group to record the group’s answers. An alternate strategy would be to “jigsaw” the questions and ask each group to answer just one of the questions. Lesson 1: A Brain Wreck 5. As a class, discuss the information that was recorded by each group. It may be helpful to ask each group to appoint a spokesperson and to allow each group to speak in turn until no more new ideas are added to the discussion. Record all answers in a visible place (i.e. chalkboard, whiteboard, butcher paper). 8 6. Keep in mind that this discussion is to elicit what students collectively know about disease and to get them thinking about the task that a scientist might face when confronted with a new disease. Keep the discussion open and encourage students to ask questions of each other. See the key below for possible responses to the questions. Prying Into Prions 7. Do not erase or throw away the responses to the questions. The students can look back on these responses as they move on to Lesson Two: Pathological Proteins? Assessment Student discussion about the causes, transmission, and treatment of disease serves as an assessment for this engagement activity. Extensions Students may want to discuss or review Koch’s postulates, a four-step procedure for identifying specific pathogens (diseasecausing agents): 1. The pathogen must be found in an animal with the disease and not in a healthy animal. 2. The pathogen must be isolated from the sick animal and grown in a laboratory culture. 3. When the isolated pathogen is injected into a healthy animal, the animal must develop the disease. 4. The pathogen should be taken from the second animal and grown in a laboratory culture. The cultured pathogen should be the same as the original pathogen. Key Student Activity Page: Molecules and Cells All items on the list contain molecules. The following items contain cells: apple, mouse, bacteria, human, lettuce, liver, and donut. (Some components contain cells, such as the wheat flour. Other components are just molecules, such as salt). Items that are part of the mouse include protein, fat, and liver. Bacteria could also be placed on the list, since most multicellular organisms contain bacteria. Mice eat apples, donuts, and lettuce to get nutrients such as vitamin C, fat, and protein that the body needs. If students describe fat (lipid) or protein as containing cells, try to clarify that they are molecules contained in cells. Particularly with advertisements for things such as liposuction or thigh cream, many may think that fat is a cell. Also, because students eat certain food items to get protein, they may think that some cells are just protein cells. In reality, all cells contain some amount of fat (lipid) and protein molecules. Student Pages: A Brain Wreck 1. What is disease? A disease might be anything that prevents a cell, tissue, organ, or organ system from working normally. 2. What causes disease? Most students will say that germs cause disease. Others will be more specific and mention bacteria, viruses, and hopefully fungi, parasites such as protozoa and “worms” such as ringworms, tapeworms, and flatworms. Hopefully students will mention genetic diseases such as cystic fibrosis or sickle cell anemia. It may be a stretch, but hopefully some students will mention aging, and environmental toxins (this could include lifestyle choices such as diet or drug use) as a source of disease. 3. Can you have an infection without having a disease? The idea that infections don’t necessarily cause disease may be tough conceptually for students and could result in an interesting class debate. Most of us contain microbes and micro-organisms throughout our bodies. Some of these are beneficial—like the bacteria in our gut that helps us digest certain foods. If being infected—that is, having another organism invade or go inside our bodies—always caused disease, we’d be in big trouble. 5. Are infectious diseases the same as transmissible diseases? This could also be 6. How can diseases be transmitted from one organism to another? In other words, how many ways can you think of to transmit an infectious disease? High school students can usually think of most of the ways that pathogens are transmitted, including: direct contact (bites, shaking hands, exchange of body fluids, etc.), air, water, and food. 7. What makes an organism immune or susceptible to a disease? Hey, it’s a great question. Some of the world’s finest minds are still working on answers. Maybe one of your students will be some day be one of them. It is known that many organisms develop immunity to a disease once they recover from the disease. It is known that non-virulent strains of some viruses can be used to create vaccines to make organisms immune to that virus. There also seems to be a genetic predisposition to some diseases, and scientists are working on various “gene therapies” to prevent or treat these diseases. This question was included because it is one that has been looked at extensively by scientists investigating transmissible spongiform encephalopathies. 8. The scientists who began studying TSEs had only the small amount of information about each disease that was presented in the reading. If you were in their shoes, how would you figure out what was going on? Answers will vary. Encourage as many ideas as possible. Do not dismiss any ideas. Ideally, write the class answers in a visible place to look back upon when students move on to Lesson 2. Essentially, the scientists looking into TSEs acted as “detectives” to track down the cause or causes of the new diseases. Students may suggest looking for common locations where the disease appeared, to check to see if something in the environment caused the illness. Some might suggest looking for other things that the victims may have in common, from diet to genetic similarity. Prying Into Prions Lesson 1: A Brain Wreck 4. Are infectious diseases preventable? In a perfect world, where science could identify every pathogen and a way to defeat each of them, infectious diseases might be preventable. This question has no correct, or “absolutely certain,” answer but allows for great discussion. an interesting discussion. This could be argued either way. If someone has a tapeworm infection, can he/she transmit it to another person? 9 Lesson 1 Student Page Molecules And Cells Circle items on this list that contain molecules and underline those items that contain cells. Apple Fat Protein Vitamin C Mouse Lettuce Bacteria Chlorophyll Human Liver Deer Mouse Donut Explain your choices. ____________________________________ ____________________________________ ____________________________________ ____________________________________ ____________________________________ ____________________________________ Lesson 1: A Brain Wreck ____________________________________ ____________________________________ ____________________________________ ____________________________________ ____________________________________ ____________________________________ ____________________________________ ____________________________________ ____________________________________ ____________________________________ 10 ____________________________________ ____________________________________ ____________________________________ ____________________________________ ____________________________________ ____________________________________ ____________________________________ Prying Into Prions Look at the list again. What items on the list are parts of the mouse? Explain. ____________________________________ ____________________________________ ____________________________________ ____________________________________ ____________________________________ ____________________________________ ____________________________________ ____________________________________ ____________________________________ What does the mouse eat? Why? ____________________________________ ____________________________________ ____________________________________ ____________________________________ ____________________________________ ____________________________________ ____________________________________ ____________________________________ ____________________________________ Student Pages A Brain Wreck A woman trembles as she stumbles forward in her thatched hut. Her unsteady gait seems strangely matched to her slurred speech. Anxious relatives sadly shake their heads, for they realize that this daughter of the Fore tribe of New Guinea will soon be dead. In a high Mule Deer Colorado meadow, a mule deer doe trembles on her wide-legged stance as her herd moves on without her. Drooling, she lowers her ears then she stumbles toward the narrow mountain brook in an effort to quench an uncontrollable thirst—a thirst that will last the remaining months of her life. A young Englishman bursts out in laughter then is overcome by a bout of crying as his parents push his wheelchair from the doctor’s office. Their physician has just explained that the most recent CAT scan shows continued deterioration in the young man’s brain and that their son’s illness is terminal. Why Learn about Transmissible Spongiform Encephalopathies? Most people cannot repeat “transmissible spongiform encephalopathies” three times quickly. Some cannot even pronounce the words once slowly! So, how are transmissible Prying Into Prions Lesson 1: A Brain Wreck In the Scottish Highlands, a ram’s head trembles slightly. He obsessively scratches and rubs against a large tree, apparently to relieve itching. He smacks his lips and bites his feet. Alarmed by a sudden noise, the ram falls down in convulsions. Can the illnesses of the New Guinea tribeswoman, the mule deer, the British teenager, and the Scottish sheep be related? In a broad sense, it appears so. All are degenerative disorders of the central nervous system. Although each illness has a different name—kuru, chronic wasting disease (CWD), variant Creutzfeld-Jakob disease (vCJD), or scrapie, microscopic examination reveals a characteristic sponge-like appearance in the brain tissue of each victim. They are just three of many diseases called transmissible spongiform encephalopathies or TSEs. These diseases are transmissible because they are capable of being transferred from one animal to another, spongiform because they cause the brain to look like a holeridden sponge under a microscope, and encephalopathies because they are diseases of the brain (pathy is Greek for disease; encephalo is Greek for brain). 11 spongiform encephalopathies transmitted? Why should you care? Why should the Colorado Division of Wildlife care? Let’s start with that last question. The Colorado Division of Wildlife (DOW) is the state agency responsible for protecting and managing wildlife and habitat, and providing wildlife related recreation. The DOW employs trained biologists and other specialists to “manage” wildlife—to use scientific knowledge to maintain healthy populations of wildlife and its habitat. Chronic wasting disease (CWD) was first recognized in Colorado in 1967, in a captive research herd of mule deer. It wasn’t until 1977 that scientists suspected that CWD was a transmissible spongiform encephalopathy, and that it might pose a significant threat to the health of Colorado’s deer and elk. Lesson 1: A Brain Wreck Protecting the state’s deer and elk was one good reason for DOW biologists to start trying to unravel the TSE mystery. But other important reasons soon presented themselves. In 1984, unusual behavior was noted among cattle on a farm in Sussex in the United Kingdom (UK or England). They wobbled and staggered and appeared fearful. Then they died. Brain samples collected from one of those cattle looked a bit like Swiss-cheese. Scientists called this new disease bovine spongiform encephalopathy or BSE. Since the original discovery, BSE has been detected in more that 182,000 cattle in more than 35,000 different herds in the UK, and in more than 3,800 cattle in other countries throughout the world! This disease seemed highly contagious, though no one knew how it spread. Could CWD be spread as quickly? 12 Sporadic Creutzfeldt-Jakob disease (CJD) has been reported for centuries. This rare disorder affects about one in a million people. Sporadic diseases like Prying Into Prions CJD occur occasionally and at random intervals in a population. Most victims are between 63 and 66 years old and die of the disease within a year of diagnosis. Autopsies show that each victim’s brain is riddled with microscopic holes. In 1996, two British teenagers and one 29 year old displayed many symptoms of classic CJD. Further research linked this new variant Creutzfeldt-Jakob disease (vCJD) of the young victims to infection with the BSE pathogen (disease causing agent). The probable cause was consumption of BSE-contaminated meat products. The “Mad Cow” scare began. Countries worldwide began screening cattle for BSE, to insure a safe food supply. Let’s get back to that question about the DOW. Deer and elk are game animals. That means that licensed hunters can harvest and eat these animals. When it was evident that BSE was zoonotic, that is, the disease could be transmitted to humans by eating meat from diseased cattle, similar concerns were raised about eating deer and elk that might be infected with CWD. The DOW has three very good reasons to study CWD. Wildlife biologists want to contain the disease, protect wild herds, and inform the public about the disease. Later, you will learn about your state wildlife agency’s efforts. Studying TSE’s—Looking for the Cause and the Cure Whenever a new or emerging disease appears, scientists try to figure out what causes the illness, if it is transmitted, how it is transmitted, and how it can be controlled or cured. If a pathogen is found, scientists then need to find out how it gets into—infects—the animal. Let’s put you in the role of one of these scientists. Consider each of these questions and write down your thoughts about each: 1. What is disease? ______________________________________ ______________________________________ ______________________________________ ______________________________________ 2. What causes disease? ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ 3. Can you have an infection without having a disease? ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ 4. Are infectious diseases preventable? ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ 6. How can diseases be transmitted from one organism to another? In other words, how many ways can you think of to transmit an infectious disease? ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ 7. What makes an organism immune or susceptible to a disease? ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ 8. The scientists who began studying TSEs had only the small amount of information about each disease that was presented in the reading. If you were in their shoes, how would you figure out what was going on? ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ Prying Into Prions Lesson 1: A Brain Wreck ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ 5. Are infectious diseases the same as transmissible diseases? 13 Educator’s Overview Lesson 2 Pathological Proteins? Duration Two 45-minute class periods (A third class period may be necessary if students have little prior knowledge of chemical bonding) Vocabulary Alpha helix (α -helix) Amino acid Amino group (-NH2 ) Summary Students read about the discovery of protein as the pathogen for TSEs and about the important roles of protein in living organisms. They construct two paper models of α -helix and β -sheet secondary protein structures. Students then create Silly Putty ®, a cross-linked polymer, and test its bounce under various temperatures. Students infer how temperature might affect the function of a protein, which is also a crosslinked polymer. Amino-terminus (N-terminus) Amyloid fibers Assay Astrocytes Beta sheet (β -sheet) Carboxyl group (-COOH) Carboxyl-terminus (C-terminus) Cross-linkage Dehydration synthesis Dipeptide Disulfide bridge Domain Fold Hydrogen group Hydrophobic Loop or turn Pathogen Peptide bond Polymer Lesson 2: Pathological Proteins? Polypeptide 14 Primary protein structure Prion Protein Quaternary protein structure R group Secondary protein structure Tertiary protein structure Prying Into Prions Learning Objectives After completing this activity, students will be able to: • Explain why an understanding of protein is essential for studying TSEs. • Identify five important roles that proteins have in living organisms. • Identify the importance of a protein’s three-dimensional shape to its role in an organism. • Identify amino acids as the building blocks of proteins and describe how they attach to one another. • Describe the four levels of protein structure or organization. • Construct paper models of α-helix and β-sheet secondary protein structures. • Create a cross-linked polymer and explore its physical properties. • Infer the effect of temperature changes on the function of protein in the body. Background Unlike all infectious diseases known at the time, the pathogen for transmissible spongiform encephalopathies or TSEs was identified as a protein. Stanley Prusiner, the scientist who isolated the protein, named it a “prion,” which is short for “proteinaceaous infectious particle.” The prion protein (PrP) exists in two forms. There is a normal cellular form (PrP c) that is found in many types of cells, including those in the central nervous system. No one knows the exact purpose of the normal prion protein, but it seems to have some role in long term memory. Then there is a pathological form (PrP res). The “res” in PrP res stands for “resistant,” because this form resists destruction! It resists “digestion” from protease enzymes in the cell, and can survive temperatures of 600ºC for more than 15 minutes. Further, it is not destroyed by some common disinfectants and is not quickly broken down by ionizing or ultraviolet radiation. The structure of proteins has always tantalized biologists. Proteins have many different functions in living organisms and each function is unquestionably linked to the molecule’s threedimensional shape. By studying the components and structures of proteins, scientists and students are better able to understand how they function normally and how some proteins with abnormal shapes can cause disease. Teaching Strategies 1. Thoroughly read the student materials for Pathological Proteins? and Silly, Silly Protein Structure. 2. Give each student a copy of the student reading pages: Pathological Proteins? 3. Give students sufficient time to read the materials or read the material as a class. 5. Tell students they will be doing a lab activity to learn more about protein folding. Explain that is it necessary to know how and why proteins fold to understand how they might misfold and cause disease. 6. Assign students to groups of four. Give each student a copy of the laboratory activity Silly, Silly Protein Structure and the activity page Polypeptide Chains. 7. Instruct the groups to attempt to complete Activities 1 and 2 of Silly, Silly Protein Structure by the end of the class period. Tell them that they would need to complete these activities at home if they are not able to complete them in class because you will need the next day’s class time to do activity 3. 8. Tell students that you will assist their group if needed. The diagrams and pictures should make these activities fairly selfexplanatory. Ask students to keep the models that they create. They may help the students’ understanding of future lessons in the module. 9. Activity 3 is a simple and enjoyable lab activity, but it does present some hazards. Please review the safety issues and precautions with students. Students should not ingest any of the lab materials. Borax® should be kept away from eyes Materials and Preparation • Student Pages: Pathological Proteins?— one photocopy per student • Student Activity Pages: Silly, Silly Protein Structure—one photocopy per student • Student Activity Page: Polypeptide Chains—one photocopy per student • Transparent tape— one roll for each group of four students • Scissors—one to four for each group of four students, depending on availability • Safety goggles— one per student • Lab aprons (if available)— one per student • Graduated cylinders or measuring cups with metric units—one per group of four students • Rulers—one per group of four students • Clear 8-oz. (or larger) plastic cups—5 for each group of four students • All-purpose white glue such as Elmer’s ® — washable glue is not as effective • Plastic spoons— 5 for each group of four students • Plastic Ziploc™ bags— one per student • Borax ®. Borax ® can be found in stores as a laundry detergent. Place one tablespoon (much more than students will need) in a Ziploc™ bag for each group of four students. • Permanent marker—one per group of four students • Water • Paper towels • Newspaper (to protect the working surface) • Access to a refrigerator/freezer or to a cold water and an ice water bath Prying Into Prions Lesson 2: Pathological Proteins? 4. Check for understanding. Ask students if they have any questions about the scientists’ discovery. Have them look at their list of “causes of disease” from Lesson 1. Did they have protein on their list of items that caused disease? Make sure students understand the terms used in the reading. If possible, you may want to demonstrate the structure of an amino acid using a model made from Styrofoam balls. Note: If your students have little knowledge of chemical structure or chemical bonding, you may need to back up a bit and spend a day making models of molecules and talking about how atoms share, gain, or lose electrons to bond with other atoms. 15 and goggles should be worn. Hands should be washed thoroughly at the end of the activity. Silly Putty ® should not be put in a sink, on carpet, or in hair. 10. After students complete the activity, review their results as a class. Be sure to relate the behavior of the Silly Putty ® polymer to protein polymers (see Key). 11. Optional: Most high school biology classes include a laboratory experiment to analyze the effect of pH and/or temperature on enzyme activity. You may wish to have students do the experiment to further reinforce the impact of protein shape on protein function. Assessment Ask students to draw the basic structure of an amino acid and label the four groups. Ask students to describe how these molecules join together to form a protein. Using diagrams and/or descriptors, ask students to depict the primary, secondary, tertiary, and quaternary structure of protein. Lastly, ask students to explain the importance of a protein’s three-dimensional shape and why a change in that shape may be important. Extensions • Students can learn more about each individual amino acid and view microscopy photos of them at Lesson 2: Pathological Proteins? http://micro.magnet.fsu.edu/ aminoacids/index.html 16 • Many protein shapes have been determined experimentally. When scientists decipher protein structures, they submit their findings to the Protein Data Bank. Students can view various protein structures at http://www.rcsb.org/ Students can enter the protein that they are interested in looking at in the search box. Prying Into Prions Key Activity 3: Silly Polymers 10. Form the Silly Putty ® into a ball. Using a ruler to measure, drop the ball from a height of 30 centimeters. How high does it bounce? Answer will vary 11. Now, place your ball in a refrigerator or ice bath for 10 minutes. Again, bounce the ball from a height of 30 centimeters. How high did it bounce this time? Why do you think this happened? Answers will vary, but the ball should bounce higher than the first drop at room temperature. 12. Now place your ball about 6 inches from a light bulb for about 5 minutes and again check how high it will bounce when dropped from a height of 30 centimeters. How high did it bounce this time? Why do you think this happened? This time, the ball will deform when dropped and may not bounce at all. 13. Now put your Silly Putty ® ball in the freezer or in an ice bath for 10–20 minutes. Check how high it will bounce when dropped from a height of 30 centimeters. How high did it bounce this time? Why do you think this happened? If frozen, the ball is likely to shatter rather than bounce. 14. Try to transfer your observations of the behavior of Silly Putty ® to the behavior of protein. Remember that proteins are the work horses of the cell or body. Why might a change in temperature, such as when a person is exposed to extreme heat or cold, cause problems? In extreme temperatures, the bonds that give a protein its shape may break and other bonds may form. The shape of the protein would change, and it would not be able to “do its job.” It is possible that the organism could die or lose important functions. Notes ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ Prying Into Prions Lesson 2: Pathological Proteins? ________________________________________ 17 Lesson 2 Student Pages Pathological Proteins? Scientists who have been studying TSEs over the past few decades have learned things that have completely challenged commonly-held beliefs about infectious diseases. Their findings will probably surprise you. Researchers looked for all the usual pathogens (diseasecausing agents). In all of the animals with TSEs, including humans, they tried to find the bacteria, virus, fungus, protozoan, or helminths (parasitic worms) responsible for this group of diseases. After painstaking and careful research, scientists did not find any of the usual causes of disease! They concluded that TSEs are not caused by bacteria or viruses. They aren’t caused by parasites. Scientists discovered that in the brain cells of each TSE victim, a normal protein had changed its shape. This abnormally shaped protein damages the brain tissue and converts other normal proteins in cells into the same abnormal shape! Lesson 2: Pathological Proteins? Prusiner’s Amazing Discovery 18 Dr. Stanley Prusiner began his medical residency in July 1972 at the University of California San Francisco in the Department of Neurology. Two months later, he admitted a female patient who was experiencing memory loss and difficulty performing some routine tasks. She was dying of Creutzfeldt-Jakob disease (CJD), thought to be caused by a “slow virus” infection. He learned that scientists were unsure if a virus was really the cause of CJD since the pathogen had some unusual properties. The disease Prying Into Prions captivated Prusiner’s imagination and he decided he wanted to research the disease. He began to read about CJD and the seemingly related diseases—kuru of the Fore people of New Guinea and scrapie of sheep. He proposed methods to study the supposed “slow virus.” Since scrapieinfected sheep were readily available, he focused his research on them. The task was daunting. The work was tedious, slow, and very expensive. He had to develop an assay—a test or procedure to purify the scrapie pathogen and determine what it was made of. Prusiner had anticipated that the purified scrapie agent would turn out to be a small virus and was puzzled when the data kept telling him that the diseasecausing agent contained protein but not nucleic acid. He named the agent a prion, which is short for “proteinaceous infectious particle.” The prion protein (PrP) can be found in two shapes. The normal form (PrP c) is found in many types of cells, including those in the central nervous system. Then there is a pathological form (PrP res). The “res” in PrP res stands for “resistant,” because this form resists destruction! It resists “digestion” from protease enzymes in the cell, it survives temperatures of 600ºC for more than 15 minutes, it cannot be destroyed by some common disinfectants or even by ionizing or ultraviolet radiation. Proteins were never thought to be infectious on their own. If you look back on your class discussion of disease, protein probably was not listed as a cause of disease. It definitely wasn’t on the list of the scientists when they began studying TSEs. The idea that proteins could be pathological (disease-causing) was counter to everything that scientists knew or believed at the time. How can proteins transmit disease? How can these diseases be treated or prevented? These questions are among the most challenging and interesting in medicine today. What are Proteins? Life would be impossible without proteins. As enzymes, proteins are the driving force behind all biochemical reactions in living things. As structural elements, they make up bones, muscles, hair, skin, and parts of all cells. As antibodies, proteins recognize invading elements and allow the immune system to get rid of the intruders. As hormones, they regulate growth and body processes. Other proteins transport nutrients into the cell and move waste out. You might say that proteins are the workhorses of life— they are how life gets things done. Just like Proteus, proteins are capable of assuming many forms. Why is the shape of a protein so important? The shape of each protein gives it a unique function. Hemoglobin’s shape lets it carry Before any protein can carry out its function, it must take on its particular shape, known as a fold. What happens if proteins don’t fold correctly? Diseases such as BSE, scrapie, Creutzfeldt-Jakob disease, CWD, Alzheimer’s, an inherited form of emphysema, and even many cancers are believed to result from protein misfolding. How are proteins made? How do they fold and misfold? Some of the worlds most prominent scientists are studying this, and you are about to join them. Proteins are Polymers Proteins are polymers, very large molecules made up of smaller molecules. In Greek, the word poly means “many” and mer means something like “unit, part, component, building-block or element.” All proteins, no matter how complex, are built from hundreds or thousands of smaller molecules called amino acids. There are only 20 different amino acids. Twenty seems like a small number to make the many types of proteins that exist, but there are only 26 letters in the alphabet, and millions of books, letters, poems and songs have been written with them! Amino Acids What do amino acids “look” like and how do they link together? Like all molecules, amino acids are made of atoms. An atom of carbon is at the center of each amino acid. Carbon atoms can share four of their electrons with other Prying Into Prions Lesson 2: Pathological Proteins? Proteins are named after Proteus, an ancient Greek god of the sea, who could change himself into any shape he felt like. Proteus, also known as the “Old Man of the Sea,” could foretell the future—but only if someone could catch him. He changed form to avoid capture. Proteus often turned himself into a sea lion to sleep on the beach at night. At other times, Proteus might be a sea serpent and sink a ship. Or—he could become a huge tidal wave and wipe out a small coastal village. oxygen. Collagen’s shape makes it a good connective tissue. Insulin fits in spaces like a key, enabling it to regulate blood sugar. 19 atoms or atom groups. In amino acids, the central carbon shares electrons with four groups of atoms. Each amino acid has an identical amino group (-NH2), carboxyl group (-COOH) and a single hydrogen group. It is the fourth group, the R group, of the molecule that makes each of the 20 amino acids different from one another. Carboxyl Group H H C H O N H C R O Lesson 2: Pathological Proteins? Amino Group 20 Amino acids always link together in the same way. The link, called a peptide bond, occurs between the carboxyl group of one amino acid and the amino group of H another. When the two amino C H acids connect, N C the carboxyl group loses a R H negatively charged O OH molecule and the amino group loses a positively charged H atom—they join by losing a drop of water! This is called dehydration synthesis— making something while losing water. Other names for this H process are dehydration reaction, dehydration C H linkage, and condensation reaction. N Don’t get confused if R your teacher or textbook H uses a different name. Two molecules linked by a peptide bond are called a dipeptide. A chain of molecules linked by Prying Into Prions peptide bonds is called a polypeptide. A protein is made up of one or more polypeptide chains. H H O H C H O N C R H O Water is lost Peptide Bond H H C C O O N H C R O H Water is lost O H Student Activity Pages Silly, Silly Protein Structure Proteins must fold into their correct 3-D shape before they can do anything useful. The structure of a protein as it folds can have as many as four levels or steps of organization. Most proteins have three. Primary Protein Structure Each end (terminus) of the polypeptide chain has a name. The first amino acid in the chain has its amino group (NH2) and this end of the chain is called the amino-terminus or the N-terminus. The last amino acid to be added still has its carboxyl group (-COOH) and is called the carboxylterminus or C-terminus. We wouldn’t bother you with all these “terms” about terminuses but the front end and back end of a protein are important. The protein always starts folding at the front end. The first or primary structure is determined by the number, kind, and order of the amino acids joined together. The amino acid sequence is genetically determined (we’ll talk Beginning of an amino acid chain… more about that later). Think of this structure Amino-terminus R H as a long bead necklace or a piece of or N-terminus H spaghetti. When the C N H protein is like this, it N C C cannot do any work. H It is just a long H polypeptide chain. O R O H H H H C N C C O R N H C O H C R Carboxyl-terminus or C-terminus C O H O N Lesson 2: Pathological Proteins? O H C C N C R R …end of an amino acid chain. Prying Into Prions 21 H Secondary Protein Structure Once the amino acid chain forms, it doesn’t just randomly wad up into a twisted mass. Short sections of the polypeptide chain, called domains, fold into recognizable shapes. These shapes are the secondary structure. Primary Protein Structure Domain 1 Domain 2 The two most common secondary structures are a coiling alpha (α ) helix or a folded beta (β ) sheet. Sometimes a portion of the amino acid chain does not form a definite shape and looks just like a loop or turn in the chain. The shapes are caused by hydrogen bonding. Hydrogen bonds are weak attractions between the Domain 3 Domain 4 positively-charged hydrogen of the amino group of one amino acid and the negatively-charged oxygen of a carboxyl group of another. Whether a certain portion of the chain coils in an α-helix, folds into a β-sheet, or just loops or turns is determined by the R groups of the amino acids in the chain. Secondary Protein Structure Loop or Turn Lesson 2: Pathological Proteins? Loop or Turn 22 Domain 1 (β β -sheet) Prying Into Prions Domain 2 Domain 3 α -helix) (α Domain 4 Tertiary Protein Structure The tertiary structure determines a protein’s function. More complex folding creates a tertiary structure. As with the secondary structure, this folding is determined by the properties of each different amino acid in the chain. Lots of different kinds of interactions happen between the R groups. Some of the R groups are positively charged and form ionic bonds with R groups that are negatively charged. Some R groups are hydrophobic, they are not charged, and like oil, repel water. The parts of the amino acid chain that are hydrophobic squish into the center of the protein molecule to avoid the liquid in the cell environment. One type of chemical bond between R groups is really special. Cysteine is the only amino acid that has an R group that contains the element sulfur. The cysteine R groups can link together to form strong bonds called disulfide bridges or crosslinkages. These special bonds help form the ultimate shape of each protein. Most animals cannot live without cysteine in their diet. Tertiary Protein Structure β -sheet Loop or Turn + - α -helix Disulfide Bridge Lesson 2: Pathological Proteins? Prying Into Prions 23 Quaternary Protein Structure Not all proteins have quaternary or fourth-level structure. In this level, two or more polypeptides are joined together by many different kinds of chemical bonds to make the finished protein. These proteins are usually pretty gigantic, on a molecular scale. Lesson 2: Pathological Proteins? Quaternary Protein Structure 24 Prying Into Prions Protein Structure and Prion Diseases As mentioned before, the prion protein exists in two forms, normal (PrP c) and pathological (PrP res). The difference between PrP c and PrP res is in their tertiary structure. The change is caused when many of the α-helices in the normal protein misfold into β-sheets. Because of their abnormal shape, PrP res proteins tend to stick to each other. Over time, the PrP res molecules stack up to form long chains called amyloid fibers. Amyloid fibers are toxic to neurons (nerve cells) and ultimately kill the cells. Cells called astrocytes crawl through the brain digesting the dead neurons, leaving holes where neurons used to be. The amyloid fibers are left behind. Then, when tissue from the brain of a victim of a TSE is examined under a microscope, one can see holes, clumps of amyloid fibers, and large numbers of astrocytes, all unique features of spongiform diseases. PrP res β -sheet PrP c Lesson 2: Pathological Proteins? Prying Into Prions 25 Activity 1: Fold an α-helix 4. Circle the O atom on the carboxyl group of every fourth amino acid beginning with amino acid #4 (amino acids #4, 8, 12, and 16). The α-helix or right-handed coil shape is caused by the hydrogen bonds that form in the carboxyl and amino groups of every R H fourth amino acid. R O 3 H H C H N Materials Needed: H H C O R R O 5 H H C C N C 4 N H H C O R O H C C N C 6 N H H C N C C O R • Student Activity Page: Polypeptide Chains 5. Hold the N-terminus end of the polypeptide chain in your left hand. Using your right hand, begin a right-handed coil. • Scissors • Transparent tape Procedure: 1. Cut out the strips labeled Polypeptide Chain 1a and Polypeptide Chain 1b along the dotted lines. Using transparent tape, join (with a hydrogen bond) the H atom of the amino group in amino acid #1 to the O atom in the carboxyl group of amino acid #5. 2. Using transparent tape, join the C-terminus of Polypeptide Chain 1a to the N-terminus of Polypeptide Chain 1b. Transparent Tape R 7 H 8 R O 9 H C H H C N C C O R O H H N 10 6. Continue to coil and hydrogen bond the following atoms of the polypeptide chain: a. H atom of amino group of amino acid #5 with O atom of carboxyl group of amino acid #8. H H C N C N C C O R b. H atom of amino group of amino acid #9 with O atom of carboxyl group of amino acid #12. 3. Circle the H atom on the amino group of every fourth amino acid beginning with amino acid #1 (amino acids #1, 5, 9, and 13). R 1 H 2 R O 3 H H C H Lesson 2: Pathological Proteins? N 26 H H Prying Into Prions C C O R R O 5 H H C C N 4 c. H atom of amino group of amino acid #13 with O atom of carboxyl group of amino acid #16. N H H C C O R O H C C N 6 N H H C N C C O R 7. Congratulations, you have constructed an α-helix or righthanded coil. Activity 2: Fold a β-sheet The β-sheet also has hydrogen bonding between the amino and carboxyl groups but in this case the polypeptide chain is folded back on itself to give a flattish R H H structure. C H N H H Materials Needed: 3 H 4 R O 5 H H C C N H H R O R O C N C 4. Circle the O atom on the carboxyl group of every fourth amino acid beginning with amino acid #4 (amino acids #4, 8, 12, and 16). • Student Activity Page: Polypeptide Chains C C N H H R O O H C N C 6 C N C C O R 5. Hold the N-terminus end of the polypeptide chain in your left hand. Hold amino acid #4 in your right hand. Fold so that the O atom on the carboxyl group of amino acid #4 touches the H atom of the amino group of amino acid #1 (sideways H bond). • Scissors • Transparent tape Procedure: 1. Cut out the strips labeled Polypeptide Chain 2a and Polypeptide Chain 2b along the dotted lines. 2. Using transparent tape, join the C-terminus of Polypeptide Chain 2a to the N-terminus of Polypeptide Chain 2b. Transparent Tape R 7 8 H R O 9 H C N H H C N C C O R 10 O H H H H C N C N C C O R 3. Circle the H atom on the amino-group of every fourth amino acid beginning with amino acid #1 (amino acids #1, 5, 9, and 13). R 1 H 2 R O 3 H H N H H C C N C C O R R O 5 H H N H H C O R O H C C N C 6 N H H Lesson 2: Pathological Proteins? C H 4 C N C C O R Prying Into Prions 27 6. Continue to fold and hydrogen bond the following atoms of the polypeptide chain: a. Fold back in the opposite direction so that the O atom of carboxyl group of amino acid #8 is on top of the H atom of amino group of amino acid #5. b. Reverse the fold again and so that the O atom of carboxyl group of amino acid #12 is on top of the H atom of amino group of amino acid #9. c. Fold back in the opposite direction so that the O atom of carboxyl group of amino acid #16 is on top of the H atom of amino group of amino acid #13. Lesson 2: Pathological Proteins? 7. Congratulations, you have constructed a β-sheet. 28 Prying Into Prions Activity 3: Silly Polymers • 1 clear 8-oz. (or larger) plastic cup (which the group should label “Water”) You’ve done a lot of brain work, so its time to get silly. You are going to cross-link a polymer to create Silly Putty ®! Since there is no feasible cross-linking activity that you can do with proteins in your classroom, this silly substitute will give you an idea of how the shape and function of proteins might change when cross-linking occurs. The objective is to cross-link a polymer and observe the changes in the physical properties as a result of this cross-linking. You will also observe changes in the physical properties of your cross-linked polymer in different temperatures. • 1 graduated cylinder The glue contains a polymer called polyvinyl acetate resin. This polymer has a structure a bit like the secondary structure of protein, like long strands of spaghetti. When water is added, the glue becomes runny, and these strands separate from each other. The Borax ® acts as a cross-linker that chemically “ties together” the long strands of the polyvinyl acetate. The strands can no longer slip and slide past one another. The Silly Putty ® will be “stiffer.” The Silly Putty ® is held together by very weak intermolecular bonds, similar to the hydrogen bonds that join portions of the polypeptide chains in proteins. Like proteins, the Silly Putty ® is not “rigid” or “solid.” There is flexibility around the weak bonds and throughout the crosslinked polymer. • Access to a refrigerator/freezer or to a cold water and an ice water bath • 1 ruler • white all-purpose glue • Borax® • 1 plastic teaspoon (which the group should label “Borax® ”) • 1 permanent marker • water • paper towels • newspaper (put down first to protect the working surface) General Safety Guidelines: • Since solid Borax ® is a bleaching agent and solution will burn the eyes, goggles and aprons should be worn. • Some people are allergic to Borax® (may have skin reaction). Wash your hands after kneading the Silly Putty ® and finishing the experiment • It is important to label anything containing or used with the Borax®. Only reuse these things with Borax ®. Borax ® is very basic and will contaminate materials for a long time, even after cleaning. Materials Needed: You will work in groups of four. Each person needs: • 1 clear 8-oz. (or larger) plastic cup • 1 plastic Ziploc™ bag Each group of 4 needs: 1. Place newspaper down to protect your work surface. Put on your goggles and lab aprons. 2. Use the permanent marker to label one plastic cup “Borax ® ” and another plastic cup “Water” for the group. Label one plastic spoon “Borax ®.” • 1 clear 8-oz. (or larger) plastic cup (which the group should label “Borax ® ”) Prying Into Prions Lesson 2: Pathological Proteins? • 1 plastic teaspoon Procedure: 29 3. Use the permanent marker to write your name on your Ziploc™ bag, your individual plastic cup, and your teaspoon. 4. Fill the “Water” cup with water. 5. Use the graduated cylinder to measure 200 mL of water. Put this 200 mL of water into the cup labeled “Borax ®.” 6. In your individual clear cup, put 6 teaspoons of glue. Add 4 teaspoons of water, from the “Water” cup, to the glue. Stir. 7. Using the “Borax ®” spoon, add 1 teaspoon of Borax ® to the water in the cup labeled “Borax ®.” Stir until most of the Borax ® is dissolved. 8. Add 4 teaspoons of the Borax ® solution to your individual cup using the “Borax ® ” spoon. Count three seconds (one-one thousand, two one-thousand, and three one-thousand). Then gently stir your cup using your spoon. Stir thoroughly so that all of the glue comes into contact with the Borax® solution. 9. Observe what is happening. Take out the Silly Putty ® and play with it. Do not try to “dry“ it on paper towels or newspaper because it will stick. The Silly Putty ® will naturally dry out and become the correct consistency as you play with it. 10. Form the Silly Putty ® into a ball. Using a ruler to measure, drop the ball from a height of 30 centimeters. How high does it bounce? Lesson 2: Pathological Proteins? ______________________________________ 30 11. Now, place your ball in a refrigerator or ice bath for 10 minutes. Again, bounce the ball from a height of 30 centimeters. How high did it bounce this time? Why do you think this happened? ______________________________________ ______________________________________ ______________________________________ ______________________________________ Prying Into Prions 12. Now place your ball about 6 inches from a light bulb for about 5 minutes and again check how high it will bounce when dropped from a height of 30 centimeters. How high did it bounce this time? Why do you think this happened? ______________________________________ ______________________________________ ______________________________________ ______________________________________ 13. Now put your Silly Putty ® ball in the freezer or in an ice bath for 10 minutes. Check how high it will bounce when dropped from a height of 30 centimeters. How high did it bounce this time? Why do you think this happened? ______________________________________ ______________________________________ ______________________________________ ______________________________________ 14. Try to transfer your observations of the behavior of Silly Putty ® to the behavior of protein. Remember that proteins are the work horses of the cell or body. Why might a change in temperature, such as when a person is exposed to extreme heat or cold, cause problems? ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ 15. Wash your hands with soap and water when you have finished the experiment Prying Into Prions H O C N H R C H 2 H O C 9 N H R C H 10 H O C 1 N H R C H 2 H N H C R O C 9 N H R C H 10 Polypeptide Chain 2 b H N C R Polypeptide Chain 2a H N C R Polypeptide Chain 1b H N 1 Lesson 2: Pathological Proteins? H H C R Polypeptide Chain 1a C O C O C O C O H N H N H N H N H C R H C R H C R H C R O C 11 O C 3 O C 11 O C 3 N H N H N H N H R C H 12 R C H 4 R C H 12 R C H 4 C O C O C O C O H N H N H N H N H C R H C R H C R H C R O C 13 O C 5 O C 13 O C 5 N H N H N H N H R C H 14 R C H 6 R C H 14 R C H 6 C O C O C O C O H N H N H N H N H C R H C R H C R H C R O C 15 O C 7 O C 15 O C 7 N H N H N H N H R C H 16 R C H 8 R C H 16 R C H 8 C O C O C O C O O O H H Polypeptide Chains 31 32 Prying Into Prions Lesson 2: Pathological Proteins? Notes ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ Prying Into Prions Lesson 2: Pathological Proteins? ________________________________________ 33 Lesson 3 Duration One or two 45-minute class periods Vocabulary Anticodon Educator’s Overview We Moose Crack The Code Summary Students read an article that explains how proteins are made from genetic information in the cell—that is, protein synthesis. They then complete an activity that simulates transcription and translation in order to decode a humorous message in a “gene.” Codon Complementary base pairing Deoxyribonucleic acid (DNA) Eukaryotes Gene Genetic code Genome Messenger RNA (mRNA) Learning Objectives After completing this activity, students will be able to: • Describe the process of protein synthesis in five simple steps. • Describe and distinguish between transcription and translation. Nontranscribed strand (a.k.a Noncoding strand) • Identify the roles of DNA, mRNA, rRNA and tRNA in protein synthesis. Nucleotide • Transcribe and translate a DNA code. Ribonucleic acid (RNA) Ribosomal RNA (rRNA) Ribosome RNA polymerase Promoter site Lesson 3: We Moose Crack The Code Protein folding problem 34 Protein synthesis Transcribed Strand (a.k.a Coding Strand or Sense Strand) Transcription Transfer RNA (tRNA) Translation Triplet Prying Into Prions hereditary information of an organism. Each gene—unit of heredity—in the genome is a sequence of DNA (deoxyribonucleic acid) that codes for one unique protein. DNA a polymer made up of four different nucleotides. Each nucleotide consists of a 5-carbon deoxyribose sugar, a phosphate group, and a nitrogen base. The sugar and phosphate group are identical for each nucleotide, but there are four different bases: adenine, thymine, guanine, and cytosine—abbreviated A, T, C, and G. DNA Nucleotide Background Understanding how proteins are made in the cell and how they fold is essential to understanding how proteins might misfold and cause disease. This activity is designed to demonstrate how the genetic code contained in DNA determines the type of proteins that the organism produces. It is an overview of the process of protein synthesis. Each protein is made using the genetic information stored in the genome, the entirety of PO4H 5’ 4’ C 1’ O C Sugar C C 3’ OH Nitrogen Base C 2’ Each carbon of the sugar is numbered. The phosphate group is attached to the 5' carbon atom, and the nitrogen base is attached at the 1' carbon. The DNA base Cytosine (C) Guanine (G) Thymine (T) Adenine (A) RNA base joins joins joins joins with with with with Guanine (G) Cytosine (C) Adenine (A) Uracil (U) The resulting three letter or three nucleotides of the mRNA that carry the genetic code are called codons. The second step of making protein is called translation. This is the step where nucleic acid “language” is converted to protein “language.” After the RNA nucleotides are joined to form an mRNA molecule, the mRNA leaves the cell nucleus and connects to a ribosome (made up of ribosomal RNA-rRNA) in the cytoplasm. Translation begins at the START codon of the mRNA. Since the codon or genetic code for each amino acid of a protein is three nitrogen bases long, molecules of transfer RNA (tRNA) with a complementary three-letter code (or anticodon) attach to the mRNA codon in sequence. One side of the tRNA molecule carries a specific amino acid which link together in that sequence, making the protein. When a STOP codon is reached, the protein molecule is complete. Teaching Strategies 1. Thoroughly read the student materials for We Moose Crack The Code and Decode That Gene. 2. Prior to class, make all photocopies. Separate the ‘genes’ on the sheets Gene Sequences A through DD so that you will be able to give one gene strip to each student. Cut apart the tRNA START molecule from the tRNA “Word” Molecules sheet so that you can give each student a tRNA START molecule. 3. Place the tRNA “Word” Molecules sheets and scissors at four stations around the room. Those starting with “A” can be placed at one station, those starting with “C” at another, and Materials and Preparation • Student Pages: We Moose Crack The Code—one photocopy per student • Student Activity Pages: Decode That Gene—one photocopy per student • Gene Sequences A through DD— One photocopy per class. Each student needs one gene sequence for this activity, 30 are provided. Copy duplicated sequences as needed to accommodate class size. • tRNA “Word” Molecules—1 photocopy of each • Four distinct “stations” in the classroom where tRNA “Word” Molecules can be placed. Those starting with “A” can be placed at one station, those starting with “C” at another, and so on. • Scissors— several at each “tRNA station” • Chalkboard, whiteboard, or some other visible writing surface Prying Into Prions Lesson 3: We Moose Crack The Code phosphate of one nucleotide is then bonded to the 3' carbon of the next nucleotide to form a DNA strand. The complete DNA molecule has two complementary strands that coil anti-parallel to each other and form a double helix. In DNA molecules, C always pairs with G, and A always pairs with T. The nucleic acid “language” of DNA is written in three letter (three nucleotide) sequences called triplets. To make a protein, this genetic information is transmitted in two stages. Each of these stages requires RNA, ribonucleic acid. RNA is very similar in chemical structure to DNA. The 5-carbon sugar in RNA is ribose instead of deoxyribose. While both DNA and RNA contain adenine (A), guanine (G), and cytosine (C) bases, the fourth nucleotide base RNA is uracil (U). There are several different kinds of RNA, each with its own function. The first step of making protein is transcription. In transcription, only one strand of the DNA double helix is transcribed. RNA polymerases are enzymes attach to the DNA at specific promoter sequences and begin constructing messenger RNA (mRNA). RNA polymerase unwinds the double helix and moves in from 5' 3' along the transcribed strand. It forms the mRNA molecule by temporarily joining complementary RNA nucleotides to the DNA nitrogen bases in this fashion: 35 so on.This will help alleviate crowding when students do their activity. 4. Give each student a copy of the Student Pages: We Moose Crack The Code. Allow students to read at their own pace, read as a group, or read aloud as a class—whatever your preference. 5. After students have read We Moose Crack The Code, reinforce the following concepts: • The building blocks of DNA and RNA are called nucleotides. • Three of the nitrogen bases of the DNA and RNA nucleotides are the same: adenine (A), guanine (G), and cytosine (C). The fourth nucleotide bases differs in the two molecules. RNA contains uracil (U) instead of thymine (T). • The DNA molecule is a double helix. The strands are complementarily paired. A always pairs with T. C always pairs with G. • Three bases of DNA will code for an amino acid. These three bases are called triplets. • mRNA forms complementary pairs with DNA. A always pairs with U. C always pairs with G. The three mRNA nucleotides that pair with the DNA triplet are called a codon. • tRNA carries an amino acid on one side and has three nucleotides called anticodons on the other to pair with the codon on mRNA • DNA strands are “read” from the 5' carbon end of the strand to the 3' carbon end of the strand. Lesson 3: We Moose Crack The Code 6. On the chalkboard or other visible place, review the example given in the students pages of base pairing and then provide additional examples and practice. 36 Molecule DNA 3-Letter Code Triplet Complementary Bases mRNA Codon Anticodon C Ë Ë tRNA G C G C G T A U A U A 7. Give each student a copy of the Student Activity Pages: Decode That Gene, one gene, and one tRNA START molecule. Tell each student that they have their own unique DNA Prying Into Prions gene and they will need to transcribe and translate that gene to get their unique message. 8. Students may require help to begin the activity. It may be helpful to begin transcription with the DNA triplet TAC as a class. Be sure that all students have identified the correct start sequence and are working from top to bottom on the right side of their gene strip. 9. While each message is humorous, each message should make grammatical sense, so any nonsensical messages are due to errors in transcription and/or translation. 10. When students have completed the activity, emphasize the importance of having a START and STOP signal in protein synthesis. Remind students that chromosomes—the long strands of DNA in their cells—contain thousands of genes. Each cell has the organism’s entire DNA, but not all of it needs to be “expressed.” For example, liver cells do not need to produce the protein for eye color. Since not all proteins need to be made at once, and some not at all, there needs to be a signal to produce a specific protein. 11. Also emphasize the problem of mutation— the addition, deletion, or change of a DNA nucleotide. The end product, in this case, the student’s message may not make sense. In a cell or organism, the change may cause death or disease. Assessment Student should be able to write a few paragraphs describing protein synthesis and the processes of transcription and translation in their own words. They should be able to explain the role of DNA, mRNA, and tRNA in protein synthesis. Extension • You may wish to introduce students to the Human Genome Project, and discuss its importance to the study of biology. Key 1. Your instructor will give you a “gene,” a portion of the DNA molecule. Write down the letter(s) that identify your gene: varies 2. If you look at your gene, what direction would the RNA polymerase enzyme move if it were reading the left side of your gene? Bottom to top. 3. What direction would the RNA polymerase enzyme move if it were reading the right side of your gene? Top to bottom. 5. What “word” did the final codon code for? Why is this codon necessary? STOP is necessary because the DNA strand has many genes and the RNA polymerase enzyme needs a signal to stop making a mRNA strand. 6. How does your message compare to those of your classmates? It is different. 7. Looking at your assigned gene, how might your message change if the fifth nucleotide base of the DNA strand after the TAC START triplet were changed? A change in one letter may change the word that is coded for. The message might change and it might not make any sense. 8. Looking at your assigned gene, how might your message change if the fifth nucleotide base of the DNA strand after the TAC START triplet were deleted? A deletion of one letter may change the word that is coded for, as well as all the words that follow. The message might change and it might not make any sense. 9. In this activity, DNA coded for a message. In the actual genetic code, each triplet codes for a specific amino acid. If this DNA strand was coding for an amino acid chain, a protein, and a nucleotide base was changed or deleted, what might happen? Any change in the DNA might change the amino acid that is coded for, so the protein that is made may not be the same. 10. What might scientists call a change in the DNA strand (change, addition, or deletion of a nucleotide base)? They might call it a mutation. Prying Into Prions Lesson 3: We Moose Crack The Code 4. What does your “message” say? A. Start A moose is my best friend Stop B. Start My large brain is my best feature Stop C. Start My best friend has the biggest moose Stop D. Start I have the biggest brain Stop E. Start I have the smallest brain Stop F. Start My best friend has the smallest brain Stop G. Start My best feature is my large brain Stop H. Start My brother has a large moose Stop I. Start I have the brain of a moose Stop J. Start My best friend is a large moose Stop K. Start My moose has the biggest mouth Stop L. Start A large moose is my best friend Stop M. Start My friend has the brain of a moose Stop N. Start My large mouth is my best feature Stop O. Start I have the best feature of a moose Stop P. Start My sister has the brain of a moose Stop Q. Start My brother has the brain of a moose Stop R. Start My sister has the best feature of a moose Stop S. Start My moose has the biggest brain Stop T. Start The friend of my sister is a moose Stop U. Start The large moose has the smallest brain Stop V. Start My sister has the smallest mouth Stop W. Start My moose has the smallest mouth Stop X. Start I have the biggest mouth Stop Y. Start I have the smallest brother Stop Z. Start I have the smallest mouth Stop AA. Start My sister is the best friend of a moose Stop BB. Start I have the smallest sister Stop CC. Start My moose is the smallest Stop DD. Start My brother is the friend of a moose Stop 37 38 Prying Into Prions Lesson 3: We Moose Crack The Code Gene Sequences A B C D E Lesson 3: We Moose Crack The Code Prying Into Prions 39 40 Prying Into Prions Lesson 3: We Moose Crack The Code Gene Sequences F G H I J Lesson 3: We Moose Crack The Code Prying Into Prions 41 42 Prying Into Prions Lesson 3: We Moose Crack The Code Gene Sequences K L M N O Lesson 3: We Moose Crack The Code Prying Into Prions 43 44 Prying Into Prions Lesson 3: We Moose Crack The Code Gene Sequences P Q R S T Lesson 3: We Moose Crack The Code Prying Into Prions 45 46 Prying Into Prions Lesson 3: We Moose Crack The Code Gene Sequences U V W X Y Lesson 3: We Moose Crack The Code Prying Into Prions 47 48 Prying Into Prions Lesson 3: We Moose Crack The Code Gene Sequences Z AA BB CC DD Lesson 3: We Moose Crack The Code Prying Into Prions 49 50 Prying Into Prions Lesson 3: We Moose Crack The Code my A A C A A C A A C A A C A A C A A C A A U Phe Phe A A U A A U A A U A A U A A U Phe Phe Phe Phe Phe Phe A A A A A A I A A A A A G A A G I Phe A A G I Phe A A G I I Phe A A G I I A A G I AAA A A G Leu Phe Phe Phe Phe Phe A A A A A A A A A A A A A A A Prying Into Prions Lesson 3: We Moose Crack The Code tRNA “Word” Molecules A A G Leu I A A U Leu I A A U Leu I Leu I Leu I Leu I Leu mymy my A A C my my A A C my my Leu my my Leu my my Leu my my Leu my Leu I my Leu my Leu I AAC AAU AAG Leu º Cut apart on red lines only 51 52 Prying Into Prions Lesson 3: We Moose Crack The Code brother brother brother brother brother brother brother brother ACC Trp Trp Trp Trp Trp Trp Trp Trp STOP Stop A C U A C U A C U A C U A C U A C U A C U A C U feature STOP Stop feature STOP Stop feature Stop feature Stop feature Stop feature Stop feature STOP A C C STOP A C C STOP A C C STOP A C C STOP A C C Cys Cys Cys Cys Cys Cys Cys Cys feature feature A C G feature A C G feature A C G feature A C G feature A C G feature A C G feature A C G Cys Cys Cys Cys Cys Cys Cys Cys A C A A C A A C A A C A A C A A C A A C A A C A Prying Into Prions Lesson 3: We Moose Crack The Code ACA A C C Stop A C G tRNA “Word” Molecules A C C feature ACG ACU A C C º Cut apart on red lines only 53 54 Prying Into Prions Lesson 3: We Moose Crack The Code the A G C A G C A G C A G C A G C A G C A G U A G U Ser Ser A G U A G U A G U A G U A G G A G G A G G A G G A G G the A G G the Ser the Ser the Ser the Ser the Ser the Ser the AGA A G G Ser Ser Ser Ser Ser Ser Ser Ser Ser A G A A G A A G A A G A A G A A G A A G A A G A Prying Into Prions Lesson 3: We Moose Crack The Code tRNA “Word” Molecules A G G Ser the A G U Ser the A G U Ser the Ser the Ser the Ser the Ser the the A G C the the A G C the the Ser the the Ser the the Ser the the Ser the Ser the the Ser the Ser the AGC AGU AGG Ser º Cut apart on red lines only 55 56 Prying Into Prions Lesson 3: We Moose Crack The Code STOP A U C A U C A U C A U C A U C A U C A U U A U U A U U A U U Tyr Tyr A U U A U U A U U Tyr Tyr A U G A U G A U G A U G A U G sister sister sister A U G Tyr Tyr Tyr Tyr A U A A U A A U A A U A sister Tyr sister Tyr sister Tyr sister Tyr sister AUA A U G Stop Tyr Tyr Tyr Tyr A U A A U A A U A A U A Prying Into Prions Lesson 3: We Moose Crack The Code tRNA “Word” Molecules A U G Stop sister A U U Stop sister Stop sister Stop sister Stop sister Stop sister Stop STOP STOP A U C STOP STOP A U C STOP STOP Stop STOP STOP Stop STOP STOP Stop STOP STOP Stop STOP Stop sister STOP Stop STOP Stop sister AUC AUU AUG Stop º Cut apart on red lines only 57 58 Prying Into Prions Lesson 3: We Moose Crack The Code is is is is is is is is Val Val Val Val Val Val Val is is is C A C is C A C is C A C is C A C is C A C is C A C Val Val Val Val Val Val Val Val C A U C A U is Val Val C A G C A G is is is Val Val Val Val Val Val Val C A G C A G C A G C A G C A A C A A C A A C A A is C A G Val is C A A C A G C A U is is Val C A U is is Val C A U is is Val is C A U is C A U C A U is CAA C A C Val Val Val C A A C A A C A A Prying Into Prions Lesson 3: We Moose Crack The Code tRNA “Word” Molecules CAG CAU C A C is CAC Val º Cut apart on red lines only 59 60 Prying Into Prions Lesson 3: We Moose Crack The Code best best best best best best best best Gly Gly Gly Gly Gly Gly best C C C best C C C best C C C best C C C best C C C best C C C best C C C best C C C Gly Gly Gly Gly Gly Gly Gly Gly C C U C C U best best best best best best Gly Gly Gly Gly Gly Gly Gly Gly C C G C C G C C G best best C C U best C C U best C C U C C U best C C U C C U best Gly Gly Gly Gly C C G best C C G best C C G C C G best C C A C C G best CCC CCU CCG CCA Gly Gly Gly Gly Gly C C A C C A C C A C C A C C A C C A C C A Prying Into Prions Lesson 3: We Moose Crack The Code tRNA “Word” Molecules Gly º Cut apart on red lines only 61 62 Prying Into Prions Lesson 3: We Moose Crack The Code brain C G C C G C Ala Ala Ala Ala Ala Ala Ala Ala brain Ala Ala C G G C G G C G G brain brain Ala brain brain Ala brain Ala brain Ala brain brain C G U brain C G U brain Ala C G U C G U brain Ala C G U C G U C G C brain C G U C G C brain C G U brain C G C brain C G C Ala Ala Ala Ala Ala Ala Ala Ala C G A C G G C G G C G G C G A C G A C G A C G A C G A C G G C G A C G G C G A Prying Into Prions Lesson 3: We Moose Crack The Code CGA CGG CGU C G C tRNA “Word” Molecules C G C brain brain Ala brain brain Ala brain brain Ala brain brain Ala brain brain Ala brain brain Ala brain brain Ala brain CGC Ala º Cut apart on red lines only 63 64 Prying Into Prions Lesson 3: We Moose Crack The Code biggest biggest biggest biggest biggest biggest biggest biggest Glu Glu Glu Glu Glu Glu Glu biggest biggest biggest C U C biggest C U C biggest C U C biggest C U C biggest C U C biggest C U C Glu Glu Glu Glu Glu Glu Glu Glu C U U C U U mouth mouth mouth Asp Asp Asp Asp C U G C U G C U G C UG mouth mouth Asp Asp Asp Asp Asp Asp C U G C U G Asp Asp Asp Asp C U A C U A C U A C U A C U A C U A C U A mouth mouth mouth C U A C U G C U G C U U mouth mouth Asp C U U mouth mouth Asp mouth C U U C U U mouth C U U C U U mouth CUA C U C Prying Into Prions Lesson 3: We Moose Crack The Code tRNA “Word” Molecules CUG CUU C U C mouth CUC Glu º Cut apart on red lines only 65 66 Prying Into Prions Lesson 3: We Moose Crack The Code my my my my my my Leu Leu Leu G A C G A C G A C G A C my my my my Leu Leu Leu Leu G A U G A U G A U my G A U my G A U my G A U Leu my G A U my G A U G A C my Leu G A C my Leu G A C my Leu G A C my my my Leu my my Leu my Leu Leu Leu Leu Leu Leu Leu Leu Leu G A G G A G G A G G A G my my my my G A G my G A G my G A G my G A G my GAC GAU GAG GAA Leu Leu Leu Leu Leu Leu Leu Leu Leu G A A G A A G A A G A A G A A G A A G A A G A A Prying Into Prions Lesson 3: We Moose Crack The Code tRNA “Word” Molecules Leu º Cut apart on red lines only 67 68 Prying Into Prions Lesson 3: We Moose Crack The Code G C A Arg Arg Arg G C U G C U G C U G C U G C U G C U G C U G C U Arg Arg G C A Pocket Moose gopher Arg Arg G C C G C C G C C G C C G C C G C C Arg Arg G C A G C A Pocket Moose gopher Arg Pocket Moose gopher Pocket Moose gopher Pocket Moose gopher Arg Pocket Moose gopher Pocket Moose gopher Arg Pocket Moose gopher Pocket Moose gopher Arg Pocket Moose gopher Pocket Moose gopher GCC Arg Pocket Moose gopher Arg Pocket Moose gopher Arg Pocket Moose gopher Arg Arg Pocket Moose gopher Arg Pocket Moose gopher G C G Arg Pocket Moose gopher G C G Pocket Moose gopher G C G Pocket Moose gopher Pocket Moose gopher G C G Pocket Moose gopher G C C Pocket Moose gopher Arg Pocket Moose gopher Pocket Moose gopher Arg Pocket Moose gopher GCU G C C Pocket Moose gopher Pocket Moose gopher Arg Pocket Moose gopher Pocket Moose gopher Arg Pocket Moose gopher Pocket Moose gopher GCG Arg Pocket Moose gopher GCA tRNA “Word” Molecules Arg Arg G C G G C G G C G Arg Arg Arg Arg Arg G C A G C A G C A º Cut apart on red lines only G C G Prying Into Prions Lesson 3: We Moose Crack The Code G C A 69 70 Prying Into Prions Lesson 3: We Moose Crack The Code friend friend friend friend Pro Pro Pro Pro Pro Pro Pro Pro G G G G G G G G A GG A Pro friend friend G G G friend G G G friend G G G G G G Pro friend Pro G G G GG U friend Pro G G G Pro G G U friend Pro Pro friend Pro friend friend Pro friend friend G G U friend G G U friend G G U friend G G U G G C friend friend Pro G G U Pro G G C friend G G C friend G G C friend G G C friend G G C friend G G C friend friend Pro Pro friend GGG Pro Pro Pro Pro Pro Pro Pro G G A G G A G G A G G A G G A G G A Prying Into Prions Lesson 3: We Moose Crack The Code GGA Pro Pro G G U tRNA “Word” Molecules Pro friend GGU G G C friend friend GGC Pro º Cut apart on red lines only 71 72 Prying Into Prions Lesson 3: We Moose Crack The Code of of of of of of of of Gln Gln Gln Gln Gln Gln Gln of G U U G U U G U U G U U G U U G U U G U U His His G U G G U G G U G G U G His His His His His G U G G U G smallest smallest G U G G U G smallest GU A His smallest G U A smallest His G U U smallest His Gln smallest His G U C of of Gln smallest Gln smallest Gln smallest Gln smallest Gln smallest Gln smallest Gln smallest of G U C of G U C of G U C of G U C of G U C smallest GUA G U C His His His His His G U A G U A G U A G U A G U A G U A Prying Into Prions Lesson 3: We Moose Crack The Code tRNA “Word” Molecules GUG GUU G U C smallest GUC Gln º Cut apart on red lines only 73 74 Prying Into Prions Lesson 3: We Moose Crack The Code START START START START START START START START UAC Met Met Met Met Met Met Met Met º Cut apart on red lines only a a a a a a Ile Ile Ile Ile Ile Ile Ile a a UAU U A C U A C U A C U A C U A C U A C U A C U A C Ile a U A G U A G U A G U A G U A G a a Ile U A G U A G U A G a a Ile a a Ile a a Ile a Ile a a a Ile a Ile a UAA Ile Ile Ile Ile Ile Ile Ile Ile Ile U A A U A A U A A U A A U A A U A A U A A U A A Prying Into Prions Lesson 3: We Moose Crack The Code tRNA “Word” Molecules UAG a U A U U A U U A U U A U U A U U A U U A U U A U 75 76 Prying Into Prions Lesson 3: We Moose Crack The Code the the the the Ser Ser Ser U C G U C G the the the UCG Ser the UCA tRNA “Word” Molecules Ser Ser Ser Ser U C G the the Ser Ser Ser U C G U C G U C G the Pocket Moose gopher Pocket Moose gopher Pocket Moose gopher Pocket Moose gopher Pocket Moose gopher Pocket Moose gopher Arg Arg U C U U C U U C U U C U U C U U C U Pocket Moose gopher Pocket Moose gopher Pocket Moose gopher Arg Arg Arg Arg Arg Arg Arg Arg Arg Ser Ser Ser Ser U C A U C A U C A U C A U C A U C A U C A U C A Prying Into Prions Lesson 3: We Moose Crack The Code Ser the U C G U C G the Pocket Moose gopher Pocket Moose gopher Arg the U C C U C C Pocket Moose gopher Pocket Moose gopher Arg the U C C U C C Pocket Moose gopher Pocket Moose gopher UCC Arg the U C C U C C U C C Pocket Moose gopher UCU Arg º Cut apart on red lines only Arg U C C U C U U C U 77 78 Prying Into Prions Lesson 3: We Moose Crack The Code has has has has has has has has UGC Thr Thr Thr Thr Thr Thr Thr Thr º Cut apart on red lines only has has has has has has has has UGU U G C U G C U G C U G C U G C U G C U G C U G C Thr Thr Thr Thr Thr Thr Thr Thr has U G G U G G has has Thr has has Thr has has Thr has has Thr has has Thr has has Thr has has Thr has UGA Thr U G G U G G U G G U G G U G G U G G Thr Thr Thr Thr Thr Thr Thr Thr U G A U G A U G A U G A U G A U G A U G A U G A Prying Into Prions Lesson 3: We Moose Crack The Code tRNA “Word” Molecules UGG U G U U G U U G U U G U U G U U G U U G U U G U 79 80 Prying Into Prions Lesson 3: We Moose Crack The Code Lys Lys Lys have have have have have have Lys Lys Lys Lys Lys Lys Lys Lys Asp large large Asp Asp Asp U U G U U G U U G large large Asp large large Asp Asp large U U G large U U G Asp large U U G large large Asp large large Asp large Asp large large Asp U U U U U U Asp Asp Asp Asp U U G U U G U U A U U A U U A U U A U U A U U A U U A U U A Prying Into Prions Lesson 3: We Moose Crack The Code UUA UUG U U U U U U U U U U U U U U U U U U tRNA “Word” Molecules U U C have U U C U U C U U C U U C have UUU U U C U U C U U C Lys have have Lys have have Lys have have Lys have have UUC Lys º Cut apart on red lines only 81 82 Prying Into Prions Lesson 3: We Moose Crack The Code START START START START START START START START UAC Met Met Met Met Met Met Met Met º Cut apart on red lines only START START START START START START START START UAC U A C U A C U A C U A C U A C U A C U A C U A C Met Met Met Met Met Met Met Met START START START START START START START START UAC U A C U A C U A C U A C U A C U A C U A C U A C Met Met Met Met Met Met Met Met START START START START START START START START UAC Met Met Met Met Met Met Met Met U A C U A C U A C U A C U A C U A C U A C U A C Prying Into Prions Lesson 3: We Moose Crack The Code tRNA “Word” Molecules U A C U A C U A C U A C U A C U A C U A C U A C 83 84 Prying Into Prions Lesson 3: We Moose Crack The Code STOP A U C A U C A U C A U U A U U A U U A U U A U U Stop Stop Stop Stop Stop Stop Stop A C U A C U A C U A C U A U U A U U A U U Stop Stop Stop A C U A C U STOP STOP Stop Stop A C U A C U STOP STOP Stop STOP STOP ACU Stop Stop Stop A C U A C U Stop Stop A C U A C U Prying Into Prions Lesson 3: We Moose Crack The Code tRNA “Word” Molecules A C U A C U A C U A C U Stop STOP Stop STOP Stop STOP Stop STOP Stop STOP Stop STOP STOP A U C STOP STOP A U C STOP STOP A U C STOP A U C STOP STOP A U C STOP Stop STOP STOP Stop STOP Stop STOP STOP Stop STOP Stop STOP STOP Stop STOP Stop STOP AUC AUU ACU Stop º Cut apart on red lines only 85 86 Prying Into Prions Lesson 3: We Moose Crack The Code Lesson 3 Student Pages We Moose Crack The Code All the TSEs (like BSE, scrapie, and CWD), cystic fibrosis, Alzheimer’s disease, and many cancers have something in common. All these apparently unrelated diseases result from protein folding gone wrong. If scientists could figure out what directs a protein to fold a certain way, they would be able to understand, treat or prevent disorders like TSEs that result from protein misfolding. For more than 50 years researchers have tried—with little success—to figure out what directs protein folding. The protein folding problem remains one of the great challenges of biology. Each day, though, scientists come closer to understanding protein folding. They use information from another code, the genetic code, which they cracked in the mid-1960s. Genes are units of genetic information and each gene provides the instructions for a single inherited DNA Nucleotides—The Genetic Alphabet Each DNA nucleotide has three parts: a phosphate group (PO4H), a 5-carbon deoxyribose sugar, and a nitrogen-containing base. The phosphate group and the sugar are identical in every nucleotide. The nucleotides differ only in their four alternative bases: adenine, thymine, guanine, and cytosine—abbreviated A, T, C, and G. DNA Nucleotide PO4H 5’ 4’ C 1’ O C Sugar C C 3’ OH Nitrogen Base C 2’ These four “letters” are the genetic alphabet. They are repeated millions or billions of times throughout a genome. A genome is the entire DNA of an Prying Into Prions Lesson 3: We Moose Crack The Code The genetic code is the set of rules by which information encoded in hereditary material in each cell of an organism makes all of the proteins needed by the organism. These proteins then determine, among other things, how the organism looks, how well its body metabolizes food or fights infection, and sometimes even how it behaves. Using the genetic code, scientists can determine a protein’s exact amino acid sequence, which brings them closer to predicting the final structure of the protein. property or characteristic of an organism—a protein. In other words, every gene makes a specific protein or amino acid chain. Genes are made of DNA or deoxyribonucleic acid. DNA is a polymer made up of four similar chemicals called nucleotides. 87 organism. The particular order of As, Ts, Cs, and Gs is extremely important. The order underlies all of life’s diversity, dictating whether an organism is a human or another species such as an amoeba, bumblebee, or squirrel. Nucleotides are joined by linking the phosphate on the 5' end of the deoxyribose sugar of one to 3' carbon of the next as shown below. When the nucleotides link together, water is lost— dehydration synthesis again! Bumblebee PO4 H , 5 , 4 C , 1 O C Sugar C C C , 3 Nitrogen Base , 2 OH PO4 H , 5 , 4 Water is lost , 1 C O C Sugar C C Red Squirrel , 3 Nitrogen Base C , 2 OH PO4 H , 5 , 4 C , 1 O C Sugar C C Lesson 3: We Moose Crack The Code , 3 88 DNA—The Double Helix The phosphate groups and the 5-carbon sugars form the backbone of each strand of the DNA polymer. The bases are joined to the sugar and stick out sideways. Notice that each carbon atom (C) of the deoxyribose sugar is numbered. Prying Into Prions OH Water is lost Nitrogen Base C , 2 Notice how the nucleotides do not join in a straight line, but stagger as they join. The causes the DNA strand to coil in a clockwise way and form a right-handed helix, just like the α-helix you made in the protein. In reality, DNA is normally found as a double stranded molecule, and the two separate strands are wound around each other as a double helix. In double stranded DNA, the bases on one strand are paired with the bases in the other strand. Adenine (A) in one strand is always paired with thymine (T) in the other and guanine (G) is always paired with cytosine (C). This kind of pairing is called complementary base pairing. If you know the base sequence of either one of , , the strands of a 3 5 DNA molecule, you A T can figure out the C G T A sequence of the other T A strand. G C G RNA—How DNA Makes Protein How does the DNA that makes up a gene code for a protein? What are the instructions? How are the instructions used to determine the order of amino acids in proteins? It’s a bit , complicated. 3 T A T A T C Bases G G C T A A T C 3. RNA is a single stranded molecule and doesn’t coil into a helix. G G C G A Roses are red, Violets are blue, DNA has A, C, G, & T But RNA needs U! 4. While there is only one type of DNA, there are several kinds and sizes of RNA. Each different kind of RNA has a different function. C G There is a simple poem you can use to remember this important fact: Yes, it’s terrible poetry, but this is a science activity! C A 2. While both RNA and DNA contain adenine (A), guanine (G), and cytosine (C), the fourth nucleotide bases differs in the two molecules. RNA contains uracil (U) instead of thymine (T). C Transcription: Making RNA T C G C G , 5 RNA is a polymer of nucleotides that is somewhat similar to DNA, but is different in many ways: 1. The 5-carbon sugar in RNA is ribose instead of deoxyribose. To make a protein, the DNA code must first be transcribed into a messenger RNA (mRNA) strand. This requires a special enzyme, RNA polymerase, which unwinds the DNA. This giant enzyme attaches to the DNA and unwinds small portions of the double helix as it moves down the transcribed strand in the 5' to 3' Prying Into Prions Lesson 3: We Moose Crack The Code When scientists first began studying genes and DNA, they found something peculiar. In eukaryotes, cells that have nucleus, the DNA always stays in the nucleus. However, protein assembly happens in the cytoplasm! In fact, even the proteins found in the nucleus are made in the cytoplasm and then transported through the nuclear membrane. How does DNA in the nucleus direct the construction of protein in the cytoplasm? The answer is RNA— ribonucleic acid. DNA instructions for protein are contained in 3-base combinations called triplets. In transcription, the chemical instructions encoded in DNA are copied to make RNA. Only one strand of the double-stranded DNA is transcribed. Logically enough, this is called the transcribed strand (although some books may call it the sense strand or coding strand). The other is called the nontranscribed strand or noncoding strand, but strangely enough, is never called the nonsense strand! 89 direction. Once DNA bases on the transcribed strands are exposed, complementary bases of mRNA will pair with them like this: DNA base C (Cytosine) G (Guanine) T (Thymine) A (Adenine) RNA base always always always always pairs pairs pairs pairs with with with with G (Guanine) C (Cytosine) A (Adenine) U (Uracil) consists of three bases that are complementary to the three bases of the codon on messenger RNA. At its other end, each tRNA carries the amino acid corresponding to the codon it recognizes. In RNA molecules, G always pairs with C and A always pairs with U. Quick Summary of Protein Synthesis Translation: Making Protein Lesson 3: We Moose Crack The Code After messenger RNA (mRNA) is formed, it leaves the nucleus and enters the cytoplasm of the cell. The next step uses the information carried by the mRNA to string together amino acids to make a specific protein. This step involves converting the nucleic acid “language”, the genetic code, to protein “language,” and that is why it’s called translation. 90 Protein synthesis is the making of protein in the cell and was described at length above. We can summarize the process in five main steps: 1. DNA provides the master genetic code (triplet) for the order of amino acids in a protein. DNA does not leave the nucleus. 2. The code is transcribed to mRNA. The complementary base pairs to the DNA triplet are called codons. During translation, the bases of mRNA are read off in groups of three, which are known as codons. Each codon represents a particular amino acid. Since there are four different bases, there are 64 possible groups of three bases, that is, 64 different codons in the genetic code. 3. mRNA carries the genetic code for the protein to a ribosome. The ribosome is made up of rRNA. Translation is carried out by a submicroscopic cellular machine called a ribosome. Oddly enough, ribosomes are made of another kind of RNA, called ribosomal RNA or rRNA. The ribosome attaches to the mRNA and moves along it, and helps to put the amino acids in the correct order. However, something is needed to get the amino acids to the ribosome to read the mRNA! Transfer RNA (tRNA) carries amino acids to the ribosome. The transfer RNA molecule has an anticodon on one end that 5. Amino acids join together through dehydration synthesis to make the protein. Prying Into Prions 4. tRNA brings the appropriate amino acid to the ribosome to assemble the protein. At the ribosome, tRNA translates the “genetic code” to “protein code.” Molecule DNA 3-Letter Code Triplet Complementary Bases mRNA Codon Anticodon C Ë Ë tRNA G C G C G T A U A U A Student Activity Pages Decode That Gene Through the processes of transcription and translation, the genetic code found in DNA creates all the proteins found in living organisms. It’s amazing how much variety in life can come from just four simple molecules. You will now be given the task of transcribing and translating a DNA message. You won’t be using the actual genetic code this time—you will work with the code for amino acids another day. Instead, you will end up with a message—words you string together using the DNA code. Getting Started 1. Your instructor will give you a “gene,” a portion of the DNA molecule. Write down the letter(s) that identify your gene: ____________________________________ 2. If you look at your gene, what direction would the RNA polymerase enzyme move if it were reading the left side of your gene? ____________________________________ If this were a real gene, there would be a portion of nucleotide bases that would identify the strand to be transcribed. This portion is called the promoter site. The RNA polymerase enzyme would attach here and the actual genetic message for a protein would start after this portion. For this exercise, you will transcribe the right side of your gene. The DNA code is written in words consisting of three letters called a codon. Once the RNA polymerase enzyme attaches to the DNA at the promoter site, it moves down along the DNA strand until it finds the START site. The START site always has the triplet of bases TAC. Carefully read your right DNA strand from the 5' carbon to 3' carbon direction and look for the DNA nucleotides TAC. Circle this start sequence on your gene! Decoding your DNA message is a two-step process. The first step, transcription, begins by pairing each DNA triplet on your gene to a complementary mRNA codon. Keeping DNA and RNA base pairing rules in mind, transcribe your mRNA molecule. Match your TAC triplet to the AUG codon of the mRNA and then complete the mRNA strand. ____________________________________ Prying Into Prions Lesson 3: We Moose Crack The Code A strand of DNA can have millions of nucleotide bases. How can you tell where a gene starts? For that matter, how can you even figure out which strand is the transcribed strand? Remember that the RNA polymerase enzyme that transcribes the DNA message into a mRNA molecule works in one direction, from the 5' carbon of one nucleotide to the 3' carbon of the next. 3. What direction would the RNA polymerase enzyme move if it were reading the right side of your gene? 91 Attach “tRNA ‘Word’ Molecules” here Lesson 3: We Moose Crack The Code Attach DNA “Gene Sequence” here. Match your circled “TAC” triplet to the complementary mRNA codon “AUG” then, transcribe the rest of your DNA by writing the complementary mRNA base on the mRNA message. 92 Prying Into Prions A U G Once your mRNA has been completely transcribed, it is time for the second step in protein synthesis—translation. You have been given sheets of tRNA molecules. Each tRNA molecule has an anticodon on one side, and a message on the other. Keeping in mind the proper way for mRNA and tRNA molecules to pair, cut out and tape each tRNA anticodon next to its complementary mRNA codon. 8. Looking at your assigned gene, how might your message change if the fifth nucleotide base of the DNA strand after the TAC START triplet were deleted? ________________________________________ ________________________________________ ________________________________________ ________________________________________ 9. In this activity, DNA coded for a message. In the actual genetic code, each triplet codes for a specific amino acid. If this DNA strand was coding for an amino acid chain, a protein, and a nucleotide base was changed or deleted, what might happen? 4. What does your “message” say? ______________________________________ 5. What “word” did the final codon code for? Why is this codon necessary? ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ 10. What might scientists call a change in the DNA strand (change, addition, or deletion of a nucleotide base)? ______________________________________ 6. How does your message compare to those of your classmates? ______________________________________ 7. Looking at your assigned gene, how might your message change if the fifth nucleotide base of the DNA strand after the TAC START triplet were changed? Lesson 3: We Moose Crack The Code ________________________________________ ________________________________________ ________________________________________ ________________________________________ Prying Into Prions 93 Lesson 4 Educator’s Overview Cannibalism, Forgetfulness, And Sleepless Nights Summary Duration One 45-minute class period Students review the process of forming proteins from DNA. They read an article that links changes in DNA—mutations—to changes in protein shape and disease. Then, using information gained from the Human Genome Project, students identify some changes in the human prion gene, PRNP, associated with inherited human prion diseases. Learning Objectives Vocabulary Allele Dominant allele Familial disease Heritable Lesson 4: Cannibalism, Forgetfulness, And Sleepless Nights • Identify and code for a mutation in the human prion gene PRNP. Homozygous • Describe the way a mutation might change the function of the PRNP gene. Human Genome Project • Describe and provide examples of a gene polymorphism. Mutation • Predict the percent of a population that might be affected by a dominant genetic disorder. Heterozygous 94 After completing this activity, students will be able to: Polymorphism Recessive allele Silent (Neutral) mutation Background This activity focuses on human prion diseases and sets the stage for upcoming activities which will focus on Chronic Wasting Disease (CWD). The reading connects the last lesson, in which students decoded a “message” from a DNA Prying Into Prions sequence, to the creation of a protein. An error in decoding, or a change in the code, can produce a protein that doesn’t “make sense.” The protein will not have the right shape to do its job. Since mutations in DNA are heritable, these altered forms of the gene can be passed to offspring. If the changed gene produces a protein that causes disease in the organism, the disease can also be passed to offspring. Since many prion diseases were thought to be genetic or familial, the human prion gene has been the focus of a lot of research. Information compiled by the Human Genome Project has enabled scientists to identify over 30 mutations in the human prion gene that are associated with human transmissible spongiform encephalopathies— ten of these mutations are linked to Creutzfeldt-Jakob disease (CJD). Some of the human prion diseases, like GerstmannSträussler-Scheinker syndrome (GSS) and Familial Fatal Insomnia (FFI), are solely inherited genetic disorders. Some, like CJD, seem to occur either spontaneously or genetically (or, as we’ll talk about in the next lesson, by exposure to abnormal prions). Another human TSE, kuru, is transmitted by eating infected tissue of a kuru victim. Students will learn how scientists apply the knowledge gained from these human prion diseases to the study of CWD in the next lesson. Teaching Strategies 1. Thoroughly read the student materials for Cannibalism, Forgetfulness, And Sleepless Nights. 2. Give each student a copy the Student Pages Cannibalism, Forgetfulness, And Sleepless Nights. Allow students to read the assignment and answer the questions silently on their own, in groups, or aloud as a class— whatever your preference. 4. Discuss the Human Genome Project and its value to the study of biology in general and to the study of prion diseases in particular. Optional: If possible, have students visit the Human Genome Project Web site at Ask students to describe these mutations, which are each associated with one or more inherited forms of CreutzfeldtJakob disease (CJD). a. b. c. d. PRNP PRNP PRNP PRNP V180I R208H E211Q M232R Answers: a. Isoleucine replaces valine as amino acid 180 in the prion protein. Isoleucine is hydrophobic, just like valine. b. Histidine replaces arginine as amino acid 208 in the prion protein. Histidine is hydrophilic and acidic and has a negative charge. Arginine is hydrophilic and basic and has a positive charge. c. Glutamine replaces glutamate as amino acid 211 in the prion protein. Glutamine is polar and moderately hydrophilic, glutamate is hydrophilic and acidic (negatively charged). d. Arginine replaces methionine as amino acid 232 in the prion protein. Arginine is hydrophilic and basic (+ charge) while methionine is hydrophobic. Materials and Preparation • Student Pages: Cannibalism, Forgetfulness, And Sleepless Nights— one photocopy per student • Optional: Provide students with access to the Internet to explore the Web site of the Human Genome Project at http://genomics. energy.gov/ Lesson 4: Cannibalism, Forgetfulness, And Sleepless Nights 3. When the assignment is completed, again emphasize that a mutation—the addition, deletion, or change of a single DNA nucleotide—can have serious consequences. Assessment http://genomics.energy.gov/ 5. Ask students to keep their copies of this lesson. They will need to refer to these pages when completing the next lesson. Prying Into Prions 95 Extensions Key • Students can research the human prion diseases: Creutzfeldt-Jakob disease (CJD), kuru, Gerstmann-Sträussler-Scheinker syndrome (GSS) and Familial Fatal Insomnia (FFI). 1. What amino acid is encoded by PRNP E200? Glutamate • Students can visit the Protein Data Bank Web site at http://www.rcsb.org/ and view the structure of normal and mutated human prion proteins. 2. What amino acid is encoded by PRNP E200K? Lysine 3. How are the chemical properties of these two amino acids different? Glutamate has a negative charge and is acidic, and lysine has a positive charge and is basic. 4. If 37 percent of the Caucasian population has two copies (are homozygous) for the PRNP M129 allele, and 12 percent are homozygous for PRNP M129V, what alleles do the other 51 percent of the Caucasian population have? The other 51 percent of the Caucasian population have one PRNP M129 allele and one PRNP M129V allele. 5. If a transmissible spongiform encephalopathy was linked to the PRNP M129V allele, what percent of the Caucasian population would be susceptible to the TSE? 63 percent Lesson 4: Cannibalism, Forgetfulness, And Sleepless Nights 6. How can the same PRNP D178N mutation be linked to two different TSE diseases and cause two different sets of symptoms? The two different PRNP 129 polymorphisms probably have different effects on the final shape of the protein. The difference in the final protein shape may affect how the protein functions in different cells in different parts of the brain. 96 Prying Into Prions Notes ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ Prying Into Prions Lesson 4: Cannibalism, Forgetfulness, And Sleepless Nights ________________________________________ 97 Lesson 4 Student Pages Cannibalism, Forgetfulness, And Sleepless Nights You discovered in the last activity that a change in one nucleotide base in the DNA sequence can result in a changed message—one that might not make sense. In an organism, a change in one nucleotide base in the DNA sequence can result in a change in the sequence of amino acids that make a particular protein. Sometimes, the result is a protein that no longer makes sense—one that does not have the right shape or fold to do its job! Any change in DNA, which is a change in genetic information, is called a mutation. The changes in the DNA are heritable, that is, the mutated form of the gene is passed to the organism’s offspring. A mutation may or may not produce a noticeable change in the protein that is made. Here’s why. Look at Table 1. The Genetic Code. There are 64 different codons in the genetic code, but only 20 different amino acids. So, some amino acids are encoded by more than one codon. For example, codons CUU, CUC, CUA, and CUG all code for the amino acid Leucine! If one nucleotide in the DNA sequence changes, but the resulting triplet still matches a codon that makes the same amino acid, the protein will not be changed and the mutation will be silent or neutral—it will do no harm. Table 1. The Genetic Code Second Base 98 First Base (5’ end) U C A G Prying Into Prions UUU UUC UUA UUG CUU CUC CUA CUG AUU AUC AUA AUG GUU GUC GUA GUG Phe Leu Leu Ile Met or Start Val C UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG A Ser UAU UAC UAA UAG Pro CAU CAC CAA CAG Thr Ala AAU AAC AAA AAG GAU GAC GAA GAG Tyr STOP STOP His Gln Asn Lys Asp Glu G UGU UGC UGA UGG CGU CGC CGA CGG AGU AGC AGA AGG GGU GGC GGA GGG Cys STOP Trp Arg Ser Arg Gly U C A G U C A G U C A G U C A G Third Base (3’ end) Lesson 4: Cannibalism, Forgetfulness, And Sleepless Nights U The Not So Silent Mutation Mutations that alter a protein may cause disease in some organisms, or increase the organism’s susceptibility to certain illnesses. To determine the effects of any mutation, one must look at both the genetic code and the characteristics of the individual amino acids that result from the change. Take a look at both Table 1. The Genetic Code and Table 2. Amino Acids and Their Chemical Properties. Let’s say that a mutation in a DNA strand resulted in an mRNA codon of CAU instead of CUU. That means that the amino acid histidine (coded for by CAU) would be placed in the amino acid chain instead of leucine (coded for by CUU). Histidine is a charged and hydrophilic amino acid that is usually found on the surface of a protein, while leucine is hydrophobic and would be found on the interior of a protein. This change would cause the protein to fold differently! Table 2. Amino Acids and Their Chemical Properties Amino Acid Abbreviation One Letter Code Chemical Properties Ala A (a) Hydrophobic Arginine Arg R (r) Hydrophilic and basic (+ charge) Asparagine Asn N ( n) Can link to a sugar; Hydrophilic Aspartate Asp D (d) Hydrophilic and acidic (- charge) Cysteine Cys C (c) Has a -SH group. Can strongly link to the -SH group of another cysteine, forming a disulfide bridge. Glutamate Glu E (e) Hydrophilic and acidic (- charge) Glutamine Gln Q (q) Moderately hydrophilic, polar Glycine Gly G (g) Small, can fit into places too small for larger amino acids Histidine His H (h) Hydrophilic and basic (+ charge) Isoleucine Ile I (i) Hydrophobic Leucine Leu L (l) Hydrophobic Lysine Lys K (k) Hydrophilic and basic (+ charge) Methionine Met M (m) Hydrophobic Phenylalanine Phe F (f) Hydrophobic Proline Pro P(p) Kinks or bends the amino acid chain Serine Ser S (s) Can link to a sugar; Hydrophilic Threonine Thr T (t) Can link to a sugar; Hydrophilic Tryptophan Trp W (w) Hydrophobic Tyrosine Tyr Y (y) Moderately hydrophilic, polar Valine Val V (v) Hydrophobic Lesson 4: Cannibalism, Forgetfulness, And Sleepless Nights Alanine Prying Into Prions 99 Can a change in just one amino acid be that big of a problem? You bet! Sickle cell disease, which most often affects those of African descent, is caused by a single change in the gene for hemoglobin, the oxygen-carrying protein in red blood cells. Only one amino acid is changed at one position in the hemoglobin molecule, distorting the normally smooth, saucershaped red blood cells into jagged sickle shapes. Another disease caused by a defect in one amino acid is cystic fibrosis. The deletion of a single amino acid in a protein called CFTR makes the protein fold incorrectly. The function of the CFTR protein is to allow chloride ions (a part of table salt) to pass through the outer membranes of cells. When this function is disrupted, glands that produce sweat and mucus are most affected. Thick, sticky mucus builds up in the lungs and digestive organs causing malnutrition, respiratory infections, and difficulties breathing. Not all properties of amino acids would cause the resulting protein to change shape, but could be harmful anyway. For example, some amino acids are designed to link to sugar molecules. These amino acids are an important part of immune system proteins. They help the cell identify foreign viruses or bacteria. Removing some of these amino acids from a protein may decrease an organism’s immunity to foreign substances, while additions of them may cause the organism to wrongly a ttack its own cells as foreign. Mutations and Prion Diseases In order for researchers to determine whether mutations had any relationship to prion diseases, they first needed to know the sequence for a “normal” prion protein. Luckily, a lot of information was available through the Human Genome Project (HGP). In 1990, the government established the Human Genome Project to compile the entire genetic sequence of humans and other organisms. The goal of the project was to: • Determine the sequences of the 3 billion chemical base pairs that make up human DNA, • Identify all the approximately 20,000–25,000 genes in human DNA, • Store this information in databases, Lesson 4: Cannibalism, Forgetfulness, And Sleepless Nights • Improve tools for data analysis 100 Scientists from around the world worked together and completed the task in 13 years. The data from the human genome is still being analyzed, and researchers are comparing the structures of different proteins from different organisms. The Human Prion Gene (PRNP) Researchers identified the human prion gene (PRNP) and the entire Prying Into Prions sequence of amino acids that are coded for by the gene. The translated portion of the gene has 253 codons, and so it codes for a protein with 253 amino acids. Scientists use a code to identify each amino acid in the sequence and also to identify any mutation in the complete prion protein. Every amino acid is numbered and identified by its single letter code. For example, all proteins, including the prion protein, begin with the amino acid methionine (met or M). So, the first amino acid in the prion protein is PRNP M1. The code PRNP D178 would mean that D, or aspartate, is the 178th amino acid in the polypeptide chain. When a mutation occurs, the single letter code for the changed amino acid follows the code for the normal form of the amino acid. For example, PRNP D178N means that N, or asparagine, has replaced the usual D, or aspartate, as the 178th amino acid in the chain. 1. What amino acid is encoded by PRNP E200? ________________________________________ 2. What amino acid is encoded by PRNP E200K? ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ Allele What? Any one form of a gene is called an allele. There are many forms of the PRNP gene. There are several forms of “normal” PRNP genes that are not associated with any prion disease. Other forms of the PRNP are linked with susceptibility to TSEs. Whether or not a person is susceptible to a TSE depends on which genes are expressed or made into proteins in the body. The dominant allele is the allele whose properties or characteristics are expressed in the organism. The recessive allele is the allele whose properties or characteristics are not expressed. It appears that alleles that are linked with susceptibility to TSEs are dominant, but there might not yet be enough data to say for sure. Each person has two alleles for every gene, including the PRNP gene, one from each parent. A person could have two normal PRNP alleles, two PRNP alleles linked to a TSE, or one of each. When a person has two of the same PRNP alleles, they are homozygous for that allele. So, a person who has two “normal” PRNP alleles is homozygous for “normal” PRNP alleles. A person who has two TSEassociated PRNP alleles is homozygous for those alleles. A person who has one normal PRNP allele and one TSEassociated PRNP allele has two different alleles and is heterozygous for the PRNP gene. Lesson 4: Cannibalism, Forgetfulness, And Sleepless Nights Approximately five to ten percent of all cases of human TSEs have been found to result from one or more mutations in the PRNP gene. One human TSE that has been extensively studied is CreutzfeldtJakob disease (CJD). Throughout the world, Creutzfeldt-Jakob disease (CJD) affects about one person in a million. Some cases appear spontaneously in elderly persons and have no known cause. It has been discovered that some victims of CJD may have any of ten identified prion mutations, but the most common is PRNP E200K. 3. How are the chemical properties of these two amino acids different? P r y i n g I n t o P r i o n s 101 4. If 37 percent of the Caucasian population has two copies (are homozygous) for the PRNP M129 allele, and 12 percent are homozygous for PRNP M129V, what alleles do the other 51 percent of the Caucasian population have? ________________________________________ ________________________________________ ________________________________________ ________________________________________ 5. If a transmissible spongiform encephalopathy was linked to the PRNP M129V allele, what percent of the Caucasian population would be susceptible to the TSE? ________________________________________ Lesson 4: Cannibalism, Forgetfulness, And Sleepless Nights Poly Who? 102 Sometimes, there are two or more sequences of DNA (and the resulting protein) that are widespread in a population. For instance, 88 percent of the Japanese population has PRNP E219 (glutamate as amino acid 219) and the remaining 12 percent have PRNP K219 (lysine as amino acid 219). The existence within a population of two or more genetically different forms of a protein is known as a polymorphism. Another common and normal polymorphism is the one described in question 4—the polymorphism of amino acid 129 in the PRNP gene. Interestingly, both the polymorphism of amino acid 219 and the polymorphism of amino acid 129 may influence susceptibility to human TSEs. A Family Affair Diseases that are inherited are called familial—they are passed on in families. The forms of Creutzfeldt-Jakob disease Prying Into Prions that can be passed through families are called familial Creutzfeldt-Jakob disease or fCJD. There are two other closely related familial human prion diseases, GerstmannSträussler-Scheinker syndrome (GSS) and Familial Fatal Insomnia (FFI). These two diseases have symptoms very similar to CJD. Like CJD, both FFI and GSS are dominant disorders—a person having one allele for the disease will get the symptoms. Familial Fatal Insomnia (FFI) is a rare prion disease that has been found in only 28 families worldwide. FFI affects the thymus, a region of the brain responsible for sleep. As the disease progresses, the patient’s insomnia worsens into complete sleeplessness. FFI is untreatable, and ultimately fatal. It appears that FFI only occurs in persons who have both the PRNP M129 polymorphism and a mutation at amino acid 178—PRNP D178N. N, or asparagine, has replaced the usual D or aspartate as the 178th amino acid in the chain. Some individuals with CJD also have the PRNP D178N mutation. These CJD victims have the M129V polymorphism. CJD affects the cerebral part of the brain and causes memory loss, jerky movements, rigid posture, and seizures. 6. How can the same PRNP D178N mutation be linked to two different TSE diseases and cause two different sets of symptoms? ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ The “Family” Dinner Sporadic CJD was first described by two German neurologists, Hans Gerhard Creutzfeldt and Alfons Maria Jakob in the early 1920s. More than a decade later, in 1936, Austrian neurologists Josef Gerstmann, Ernst Sträussler, and I. Scheinker, detailed their findings on Gerstmann-Sträussler-Scheinker syndrome (GSS). Family records of the victims of GSS showed a history or seemingly related deaths. It appeared to most doctors studying these unusual neurological diseases that most human transmissible spongiform encephalopathy diseases were either sporadic (occurring at random) or inherited. The disease that changed this perception was kuru, the “laughing death” of the South Fore tribe in New Guinea. Kuru is a progressive, fatal brain malady that robs its victims of the ability to walk, talk, and even eat. Anthropologists who studied the disease in the 1950s first thought that it was a genetic disorder, because it had a tendency to occur among family members. Most scientists did not think kuru could be solely a genetic disorder, because kuru was too common and always fatal. Then, veterinary pathologist William Hadlow observed similarities between kuru and scrapie. Both diseases caused trembling, loss of coordination and certain death. Like scrapie, kuru produced a microscopic “Swiss-cheesing” of the brain. Shirley Lindenbaum, another researcher, pinpointed a probable cause. Female villagers honored the death of close relatives—even kuru victims—by eating them. When an individual died, the female kin were responsible for the dismemberment of the corpse. The women would feed portions of the brains and various organs to their children and the elderly. In 1966, Carleton Gajdusek and Michael Alpers at the U.S. National Institutes of Health tested Lindenbaum’s idea. They transmitted kuru to chimpanzees by injecting them with infected brain tissue, proving that the cause was not genetic. They thought it was a slow virus and began focusing attention on prevention. Ritual cannibalism was outlawed. Although kuru was later found to be caused by prions, and not a slow virus, prevention has worked. Generations born after the ban are kuru-free. Older people in the region are still “getting” kuru. All of the current victims were born before 1950, meaning that the incubation period for some of the victims was over 40 years! Kuru research has been important to the study of TSEs, because it demonstrated that prion diseases can be transmitted by eating infected tissues. It also demonstrated that the disease could be transmitted to genetically similar species. Today, researchers at the Colorado Division of Wildlife and other government and private agencies are using this knowledge, and genetic research from the Human Genome Project, to find answers to questions about chronic wasting disease (CWD). Their work will be the topic of our next lesson. Lesson 4: Cannibalism, Forgetfulness, And Sleepless Nights Kuru was first noticed in New Guinea in the early 1900s and the “laughing death epidemic” reached its peak in the early 1960s. Between 1957 and 1968, over 1,100 South Fore people died from kuru. The vast majority of the victims were women. Eight times more women than men contracted the disease! It seemed to affect small children and the elderly at a high rate as well. Since scrapie was an infectious disease, Hadlow believed that kuru was also infectious. But how was the disease transmitted? P r y i n g I n t o P r i o n s 103 Educator’s Overview Lesson 5 Oh…Deer Summary Students read an article that chronicles the discovery of chronic wasting disease and the disease’s emergence as a public concern. They then compare the 14 domains of the prion protein sequence for nine mammalian species and predict the susceptibility of each species to different prion diseases. Learning Objectives Duration After completing this activity, students will be able to: One or two 45-minute class periods • Compare the prion protein sequences of nine mammalian species. Vocabulary Biopsy Prion or “protein only” hypothesis • Hypothesize the susceptibility of different species to different prion diseases and provide support for their prediction. • Evaluate the relative importance of numbers of variations in prion sequence or specific polymorphisms to predicting susceptibility to prion diseases. Background Lesson 5: Oh…Deer Chronic wasting disease, a fatal disease of deer, elk, and moose, has been found in Colorado for 40 years. It didn’t garner headlines until the mid1990s, when British health officials linked eating food products from cattle affected with bovine spongiform encephalopathy (BSE) to a new human transmissible spongiform 104 Prying Into Prions encephalopathy (TSE) called variant Creutzfeldt-Jakob disease (vCJD). The perception was that since both chronic wasting disease and bovine spongiform encephalopathy were the same type of disease, humans could potentially acquire a TSE from eating meat from deer or elk affected with chronic wasting disease. This lesson examines the types of information that scientists look at to attempt to predict transmission of prion diseases from one species to another. Students will compare the 14 domains of the prion protein sequence for nine mammalian species and predict the susceptibility of each species to different prion diseases. They will try to identify crucial portions of the prion protein to protein function, as well as identify domains that seem associated with susceptibility or resistance to prion disease. They will hypothesize whether the numbers of variations between species or specific polymorphisms are a more likely predictor of TSE transmission between species. Teaching Strategies 8. Ask students to complete their questions individually or in pairs. 1. Thoroughly read the student materials for Oh…Deer. 9. Discuss student answers in class. This allows time to really probe students’ scientific reasoning. Can students point to evidence that support their answers? Can students think of counter examples? What assumptions are they making? What might they be able to do to gather further evidence for their predictions? 2. Give each student a copy of the Student Pages Oh…Deer and Prion Amino Acid Sequences by Protein Domain. Students may also need to refer back to the reading and tables in Lesson 4 Cannibalism, Forgetfulness, And Sleepless Nights. 3. Allow students to read the article silently on their own, in groups, or aloud as a class— whatever your preference. 4. Review the concepts of domain and polymorphism. Make sure that students understand what these terms mean and how to identify them in a protein sequence. 5. This activity involves comparison of many gene sequences. You may want to assign the students to groups and have each group compare one to three of the domains, depending on the number of amino acids in each domain. Then, students can compile the data as a class. Because this comparison requires so much attention to detail, it would be wise to have at least two groups independently compare the prion sequences for each domain. Alternatively, you may want to allow additional class time or assign homework if you wish each student to complete the comparison individually. 7. After each student has completed their assigned task, compile the data table. Students’ ability to answer questions 1 through 12 using data that they have compiled from comparing prion protein sequences serves as the assessment for this lesson. Extensions • Using the same procedures used in Oh…Deer, students can compare the possibility of transmission of other mammalian spongiform encephalopathies to various species. They can examine the role of polymorphism in the transmission or susceptibility of animals to various prion diseases. There are prion protein sequences for 53 species listed by domain available at this Web site: Materials and Preparation • Student Pages: Oh…Deer—one photocopy per student • Student Activity Pages: Prion Amino Acid Sequences by Protein Domain— one photocopy per student Scissors—one per student http://www.cyber-dyne.com/ ~tom/protein_domains.html Students will need to research the scientific name of the species to use this website, but that information is readily available in books and on the Internet. Lesson 5: Oh…Deer 6. Give each student or group scissors. Tell students that the easiest way to compare prion sequences is to separate the species in each domain and compare them in the pairs requested in the table. Assessment P r y i n g I n t o P r i o n s 105 Three other mammalian transmissible encephalopathies have been linked with consuming feed containing either BSE-infected cattle or scrapie-infected sheep. They are transmissible mink encephalopathy (TME), feline spongiform encephalopathy (FSE), and exotic ungulate encephalopathy (EUE). TME is rare, and has largely been confined to the United States, although incidents have also occurred in Canada, Finland, East Germany and Russia. TME takes the form of a rapidly evolving epidemic, usually involving single mink farms, and appears not to spread from mother to offspring. The disease is fatal and appears to be associated with the feeding of contaminated food (containing BSE-infected cattle or scrapie-infected sheep) and perpetuated through cannibalism among mink. FSE was first reported at the Bristol Veterinary College in Great Britain. FSE has been found in domestic cats and captive wild cats, including tigers, puma, an ocelot and a cheetah; all fed infected feedstuffs. Currently, there is research underway in Colorado to test whether mountain lions can contract FSE from eating meat from CWD-infected deer. EUE has been seen in captive nyala, gemsbok, Arabian oryx, eland, kudu, scimitarhorned oryx, ankole, and bison. All of these animals are in the same taxonomic family as cattle. All of these animals were in zoos and were fed either BSE-infected cattle or scrapieinfected sheep. BSE prions from cattle can be experimentally transmitted to cats, mink, mice, pigs, sheep, goats, marmosets and cynomolgus monkeys. Students can also construct a timeline of events that chronicles the discovery and research findings of the various transmissible spongiform encephalopathies. Pre-GPI Alpha 3 Asn Alpha 2 Loop 3 Beta 2 Loop 2 Alpha 1 Loop 1 Beta 1 Core Octa. repeat Pre-repeat Species Compared Number of Potential Differences in Each Prion Protein Domain Signal Table 1. Comparison of Prion Protein Domain for Select Species Odocoileus virginianus & Odocoileus hemionus hemionus 0 Odocoileus virginianus & Cervus elaphus nelsoni 1 Odocoileus virginianus & Alces alces shirasi 1 1 Odocoileus virginianus & Bos taurus 2 1 1 1 2 1 5 5 Odocoileus virginianus & Homo sapiens 9 1 1 2 2 1 3 1 1 4 25 Ovis aries & Homo sapiens 9 1 1 3 2 1 1 1 2 4 25 1 1 1 1 1 1 2 1 2 1 4 22 2 1 3 1 7 25 Ovis aries & Bos taurus 1 Bos taurus & Homo sapiens 9 Mus musculus & Homo sapiens 7 1 Pan troglodytes & Homo sapiens Lesson 5: Oh…Deer 1 0 Odocoileus virginianus & Ovis aries 106 Total Number of Differences Key 1. What prion domains seem to have the most changes or variations in amino acids among the species? The domains that have a lot of variation among species include signal, pre-repeat, octa-repeat, core, loop 1, loop 2, loop 3, alpha 2, alpha 3, and pre-GPI. Prying Into Prions 1 1 1 2 1 6 1 2. Which prion domains seem to have the least changes or variations among species? Beta 1, alpha 1, beta 2, and asn domains have the least changes or variations among the species. 3. Usually, when there are few changes in amino acids in the prion domains among species, it means that those portions of the protein are most essential for protein function. Which domains seem most important in prion protein function? Beta 1, alpha 1, beta 2, and asn domains seem the most important to prion protein function. 4. Researchers hypothesize that species with similar prion proteins are more susceptible to each other’s prion diseases. White-tailed deer and mule deer are very susceptible to CWD. What other species might be more susceptible to transmission of CWD from white-tailed deer? According to this hypothesis, the North American moose prion sequence is identical to the white-tailed deer prion sequence and moose might be more susceptible to the transmission of CWD. The next most susceptible species might be the elk. 5. Check the Colorado Division of Wildlife Web site: http://wildlife.state.co.us Has CWD been found in this species (your answer to question 4)? When? In September 2005, a male moose (Alces alces) harvested on the west side of the Never Summer Range in northcentral Colorado was diagnosed with CWD. 8. Some researchers believe that cattle (Bos taurus) contracted bovine spongiform encephalopathy (BSE) after consuming meat and bone meal from scrapie-infected sheep (Ovis aries). Other scientists believe that BSE is a sporadic disease that was amplified or made worse from consuming the scrapiecontaminated feed. Comparing the data prion proteins for the two species, are either of these events likely? Explain your answer. There are only 6 differences in the polymorphisms of these prion sequences of domestic sheep and cattle, so the events seem likely. 9. Researchers also believe that humans (Homo sapiens) who have variant CreutzfeldtJakob disease (vCJD) acquired the disease from eating meat products from BSE-infected cattle (Bos taurus). Comparing the data prion proteins for the two species, is this event likely? Explain your answer. There are 22 differences in the prion protein sequences of humans and cattle, so this event seems unlikely. 10. So far, all human vCJD patients have two copies of PRNP M129 (methionine as amino acid #129 in the protein). It appears that being heterozygous or homozygous for PRNP M129V (valine as amino acid 129 in the protein) provides some resistance to getting vCJD. On what domain of the human prion does this polymorphism occur? This polymorphism occurs on the beta 1 domain of the human prion protein. 11. A recent study was conducted to determine the effect of a polymorphism on the appearance of clinical CWD symptoms in mule deer. The study compared mule deer that were homozygous for the serine (S) codon for amino acid 225 (that is the deer were 225SS) with mule deer that were heterozygous for serine (S) and Lesson 5: Oh…Deer 6. People have reported scrapie in sheep for over 300 years. Not all sheep get scrapie. Veterinarians looked at the prion gene sequences of sheep with scrapie and sheep that were resistant to scrapie. They found many polymorphisms—different forms of genes—in sheep prions for amino acids number 171 and 136. Sheep that had polymorphisms that coded for the amino acid arginine (R) as the 171st amino acid in the protein, and for alanine (A) as the 136th amino acid in the protein chain, seemed resistant to getting scrapie. However, sheep that had prion genes that coded for glutamine (Q) or histidine (H) as the 171st amino acid in the prion protein chain, or that had valine (V) as the 136th amino acid in the chain, were likely to get scrapie. In what domains of the prion protein are these polymorphisms found? These polymorphisms are found on loop 1 and loop 3. 7. So far, there is no recorded case of a human contracting a prion disease from eating meat products from domestic sheep. Comparing the prion proteins for humans (Homo sapiens) and sheep (Ovis aries), is this event likely? Explain your answer. There are 25 differences in the prion sequences of domestic sheep and humans, so this event does not seem likely. P r y i n g I n t o P r i o n s 107 phenylalanine (F) at codon 225 (deer that were 225SF). The results of the study were that the 225SS mule deer showed clinical signs of CWD before the 225SF deer did. What results would you predict for mule deer that were homozygous for phenylalanine (F) at codon 225—the 225FF deer? How could you test your prediction? Students may suggest that the appearance of clinical CWD in 225FF deer is delayed even longer or that these deer are resistant to CWD. Any study suggested should include testing deer with all three genotypes (225SS, 225SF, and 225FF). Lesson 5: Oh…Deer 12. Thinking about your answers to questions 6 through 11, which seems more important in predicting the transmission of a prion disease, the number of variations in the prion genes of the species or specific polymorphisms? Support your answer. Specific polymorphisms seem more important in predicting the transmission of a prion disease. There are 22 differences between cattle and human prion proteins, but there are some cases of humans getting vCJD from eating meat products from BSE cattle. On the other hand, humans with the PRNP M129V polymorphism seem to be resistant to vCJD. Specific polymorphisms (R171 and A136) seem to provide sheep with resistance to scrapie. So, individual polymorphisms seem to be more important. 108 Prying Into Prions 13. Human diseases are often studied using other animals, particularly mice (Mus musculus). According to your data in Table 1. Comparison of Prion Protein Domain for Select Species, which species has the closest prion protein sequence to humans? Why is the mouse used to study prion diseases instead? There is only one difference in the prion protein sequence of chimpanzees and humans, so that would be the best species to study human prion diseases. There are 25 differences in the prion protein sequences of humans and mice. However, using chimpanzees would be more expensive and more difficult than using mice for medical research because chimpanzees are larger, have longer life spans, fewer offspring, and are less numerous. Plus, many people would consider using chimpanzees unethical, because of their closeness to humans, their intelligence, and their rarity in the world. Deer Mouse Notes ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ Lesson 5: Oh…Deer ________________________________________ P r y i n g I n t o P r i o n s 109 Lesson 5 Student Pages Oh…Deer No one knows for sure where or how the disease started. It was first noticed by researchers at a Colorado State University (CSU) research facility in 1967. Captive mule deer that were being maintained for nutritional studies began to lose weight as young adults. They later became listless, started slobbering and drooling, and seemed constantly thirsty. Some stopped socializing with other deer. The affected deer died within months of the appearance of these symptoms. At first, researchers thought that the illness, named chronic wasting disease or CWD because of the deer’s symptoms, was caused from nutritional deficiencies, poisoning, or stress from confinement. However, CWD seemed highly contagious. In the 1970s, 90 percent of the deer that stayed more than two years in the research facility died or had to be euthanized. In 1980, CWD was first noticed in deer at Wyoming’s Sybille Research Unit, about 120 miles northwest of Colorado’s facility. Wyoming had sent deer to Colorado for breeding purposes. To make matters worse, elk at both facilities contracted the disease. It was becoming clear that CWD was an infectious disease, but what caused it? Lesson 5: Oh…Deer Student Discovers New Disease 110 In 1977, Dr. Elizabeth Williams, studying for her doctorate in veterinary pathology at Colorado State University, Prying Into Prions made an extraordinary discovery. She decided to look at tissue samples from the brains of some of the “wasting” deer. She saw that the tissue was full of microscopic holes. She shared her findings with Dr. Stuart Young, a neuropathologist at CSU. The holes were unmistakably like scrapie, the sheep sickness that was the first documented spongiform encephalopathy. Elizabeth Williams and Stuart Young had discovered a new transmissible spongiform encephalopathy (TSE). Scientist Proposes New Agent of Disease Before 1982, most researchers believed that TSEs were some sort of genetic disorder, or occurred spontaneously, or were caused by a slow-incubating virus. Then, Stanley Prusiner of the University of California at San Francisco proposed the prion or “protein only” hypothesis—that a misfolded protein could become a pathogenic entity that kills. Prusiner’s ideas set off a fire storm of criticism in the scientific community. At the time, no self-respecting biologist believed that a pathogen could replicate and pass on its traits without assistance from nucleic acids (DNA or RNA). While it is only natural for scientists to be skeptical of new ideas that do not fit within the accepted realm of scientific knowledge, Prusiner became the target of vicious personal attacks in the media. Still, he continued his research. It would take many years for Prusiner and others to gather enough evidence to convince the scientific community that some proteins could in fact copy themselves. By the early 1990s, the existence of prions was coming to be accepted in many quarters of the scientific community, and in 1997, Prusiner would receive the Nobel Prize in Physiology/Medicine. Once again, as throughout history, scientists who press on in spite of criticism, often contribute the most to our body of knowledge. In the Meanwhile Dr. Elizabeth Williams graduated with her doctorate in Veterinary Pathology and went to work for the University of Wyoming in Laramie. She continued researching chronic wasting disease. Michael Miller, veterinarian for the Colorado Division of Wildlife, later joined in these efforts. Initially, the disease seemed to be confined to deer and elk at Colorado’s Foothills Wildlife Research Facility and Wyoming's Sybille Research Unit. For over a decade, only veterinarians, infectious disease specialists, and neurologists knew anything about CWD and other prion diseases. The diseases didn’t grab any headlines. That would eventually change. 1985 That same year, a handful of cattle from various areas in the United Kingdom began dying of a strange illness. Examination of the dead cattle’s brains revealed abnormal, microscopic holes— the same spongy appearance of scrapie in sheep, kuru in the Fore people, and CWD in deer and elk. The new disease was named bovine spongiform encephalopathy or BSE. The “Mad Cow” Epidemic BSE quickly ballooned into an epidemic that struck more that 37,000 cattle per year by 1992. Scientists studying the human genome had identified the human prion gene, and many prion genes for other species. They proposed a cause of the new illness. Feed producers in Britain used parts of dead animals to boost protein content in their products. Body parts of sheep that had died from scrapie were used for cattle feed. By the time the full extent of the epidemic was known, many BSE-infected cattle entered the human food chain. In fact it has been estimated that 840,000 to 1.25 million infected cattle became hamburger or roast or other meat products. British health officials, concerned that the transmission of BSE to humans was a possibility, increased surveillance of Creutzfeldt-Jakob disease (CJD). CJD normally occurs in older people, but in 1996 a few teenagers in the United Kingdom started showing CJD symptoms. Biopsies (the taking of tissue for examination) of brain tissue of these Lesson 5: Oh…Deer By 1985, a few cases of CWD had been diagnosed in free-ranging deer and elk. Researchers began to wonder whether the disease originated in the wild and spread to the captives, or vice versa. The original stock for both research facilities came from the wild. The wild and captive populations had plenty of time to mingle. Wild deer and elk often lingered around captives, especially during the mating season. Either way, state wildlife officials were concerned about maintaining the health of the wild herds. P r y i n g I n t o P r i o n s 111 victims revealed that a new variant, vCJD, involved the same prion strain as seen in BSE cases in cattle. Newspapers, television stations, and radio programs all carried the story. Panic set in worldwide. Hundreds of thousands of cattle were put down. Oh Deer, Oh Deer, Oh Deer! With the discovery of BSE transmission to humans, chronic wasting disease and other prion diseases came under the microscope. Since both chronic wasting disease and BSE are transmissible spongiform encephalopathies, and if cattle affected with BSE could cause new variant CJD, then why couldn’t eating meat from deer or elk affected with chronic wasting disease cause human disease? Could CWD spread to other wildlife species? Could CWD spread to livestock? There were so many questions. Elizabeth Williams and Michael Miller, officials at the federal Centers for Disease Control and Prevention, and at the Colorado Department of Health, worked to find answers. humans. This is a brief list of some of the facts known about prion diseases in 1996: A. Some prion diseases seem to arise spontaneously. B. Prion disease can be transmitted by eating infected tissue. Kuru victims contracted the disease by eating tissue from deceased relatives. Cattle may have contracted BSE by eating tissue from scrapie-infected sheep. Humans probably contracted vCJD by eating tissue from BSE-infected cattle. C. Prion diseases can be transmitted to genetically similar species. Chimpanzees were easily infected with kuru from human victims. D. Prion diseases can be transmitted to genetically-dissimilar species under certain circumstances. Cattle fed feed containing sheep parts developed BSE, some humans that consumed BSEcontaminated meat developed vCJD. E. In the hundreds of years that scrapie has been infecting sheep, it appeared that no human had acquired a prion disease by eating sheep meat. F. Sheep with scrapie were contagious to other sheep, and seemed to transmit the disease through contact with each other. G. Prions were hard to destroy. Normal sterilization methods that eradicate bacteria and viruses did not work. Lesson 5: Oh…Deer First Steps 112 The first step that scientists take when trying to find answers to new questions is to compile all the research that has ever been done on the topic. The researchers that were concerned that CWD might be transmissible to other species, especially to humans, gathered together all known research on prion diseases in animals and Prying Into Prions Comparing Prion Sequences Later, researchers would compile and compare the amino acid sequences produced by the prion genes from as many species as possible. Each sequence was presented in a standard manner, according to the human prion (PRNP) sequence, so that researchers could easily compare the similarities and differences between species. By 2003, many sequences for the deer family had been sorted out. As in humans, many polymorphisms of the prion gene were found in each species. Protein Domain. The amino acids in each sequence are represented by their one letter code. The 14 domains of the prion sequences are arranged in order and the number in parentheses before each domain indicates the starting amino acid of the sequence. Some species have a The researchers compared the prions greater or lesser number of amino acids by protein domain. Remember that a in each domain. When a species doesn’t domain is a segment of the polypeptide have an amino acid in a certain place in chain that folds into a recognizable shape. the sequence, the space is marked with a dash (-) so that the domains of each species can be easily Secondary Protein Structure compared. Loop or Turn Domain 1 (β β -sheet) Domain 2 Domain 3 (α α -helix) Your instructor will tell you how many prion domains you or your group will compare. Compare the prion domains for two species at a time. Count the number of differences in the amino acids in each domain and enter your answer Domain 4 in the correct row and column of Table 1. Comparison of Prion Protein Domain for Select Species. The prion protein has 14 domains. The first domain, the signal sequence, contains Example: If you compare the “signal” the “start” code and also identifies the domain for white-tailed deer and humans, final destination of the prion protein in the you would find 9 differences: cell. The last domain, the pre-GPI, adds Species (1) signal a fat molecule to the prion protein to anchor it to the cell membrane. Odo.vir m v k s h i g s w i l v l f v amw s d v g l c Hom.sap m a n l - - g c wm l v l f v a t w s d l g l c Lesson 5: Oh…Deer You are now going to take on the role of the CWD researcher and compare the prion sequences from nine species. Keep in mind that you will only be comparing one of the prion polymorphisms of each species. You will be given Prion Amino Acid Sequences by P r y i n g I n t o P r i o n s 113 Pre-GPI Alpha 3 Asn Alpha 2 Loop 3 Beta 2 Loop 2 Alpha 1 Loop 1 Beta 1 Core Octa. repeat Pre-repeat Species Compared Number of Potential Differences in Each Prion Protein Domain Signal Table 1. Comparison of Prion Protein Domain for Select Species Total Number of Differences The answer “9” has been placed in the correct box in Table 1. Comparison of Prion Protein Domain for Select Species for you. Odocoileus virginianus & Odocoileus hemionus hemionus Odocoileus virginianus & Cervus elaphus nelsoni Odocoileus virginianus & Alces alces shirasi Odocoileus virginianus & Ovis aries Odocoileus virginianus & Bos taurus Odocoileus virginianus & Homo sapiens 9 Ovis aries & Homo sapiens Ovis aries & Bos taurus Bos taurus & Homo sapiens Mus musculus & Homo sapiens Pan troglodytes & Homo sapiens After you have completed the table, answer these questions: 1. What prion domains seem to have the most changes or variations in amino acids among the species? ________________________________________ ________________________________________ ________________________________________ ________________________________________ 2. Which prion domains seem to have the least changes or variations among species? Lesson 5: Oh…Deer ________________________________________ ________________________________________ ________________________________________ ________________________________________ 114 3. Usually, when there are few changes in amino acids in the prion domains among species, it means that those portions of the Prying Into Prions protein are most essential for protein function. Which domains seem most important in prion protein function? ________________________________________ ________________________________________ ________________________________________ ________________________________________ 4. Researchers hypothesize that species with similar prion proteins are more susceptible to each other’s prion diseases. White-tailed deer and mule deer are very susceptible to CWD. What species might be more susceptible to transmission of CWD from white-tailed deer? ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ 5. Check the Colorado Division of Wildlife Web site: http://wildlife.state.co.us Has CWD been found in this species (your answer to question 4)? When? ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ 6. People have reported scrapie in sheep for over 300 years. Not all sheep get scrapie. Veterinarians looked at the prion gene sequences of sheep with scrapie and sheep that were resistant to scrapie. They found many polymorphisms—different forms of genes—in sheep prions for amino acids number 171 and 136. Sheep that had polymorphisms that coded for the amino acid arginine (R) as the 171st amino acid in the protein, and for alanine (A) as the 136th amino acid in the protein chain, seemed resistant to getting scrapie. However, sheep that had prion genes that coded for glutamine (Q) or histidine (H) as the 171st amino acid in the prion protein chain, or that had valine (V) as the 136th amino acid in the chain, were likely to get scrapie. In what domains of the prion protein are these polymorphisms found? ________________________________________ ________________________________________ ________________________________________ 7. So far, there is no recorded case of a human contracting a prion disease from eating meat products from domestic sheep. Comparing the prion proteins for humans (Homo sapiens) and sheep (Ovis aries), is this event likely? Explain your answer. ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ 9. Researchers also believe that humans (Homo sapiens) who have variant Creutzfeldt-Jakob disease (vCJD) acquired the disease from eating meat products from BSE-infected cattle (Bos taurus). Comparing the data prion proteins for the two species, is this event likely? Explain your answer. ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ 10. So far, all human vCJD patients have two copies of PRNP M129 (methionine as amino acid #129 in the protein). It appears that being heterozygous or homozygous for PRNP M129V (valine as amino acid 129 in the protein) provides some resistance to getting vCJD. On what domain of the human prion does this polymorphism occur? ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ Lesson 5: Oh…Deer ________________________________________ ________________________________________ ________________________________________ ________________________________________ 8. Some researchers believe that cattle (Bos taurus) contracted bovine spongiform encephalopathy (BSE) after consuming meat and bone meal from scrapie-infected sheep (Ovis aries). Other scientists believe that BSE is a sporadic disease that was amplified or made worse from consuming the scrapiecontaminated feed. Comparing the data prion proteins for the two species, are either of these events likely? Explain your answer. P r y i n g I n t o P r i o n s 115 11. A recent study was conducted to determine the effect of a polymorphism on the appearance of clinical CWD symptoms in mule deer. The study compared mule deer that were homozygous for the serine (S) codon for amino acid 225 (that is the deer were 225SS) with mule deer that were heterozygous for serine (S) and phenylalanine (F) at codon 225 (deer that were 225SF). The results of the study were that the 225SS mule deer showed clinical signs of CWD before the 225SF deer did. What results would you predict for mule deer that were homozygous for phenylalanine (F) at codon 225—the 225FF deer? How could you test your prediction? ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ 12. Thinking about your answers to questions 6 through 11, which seems more important in predicting the transmission of a prion disease, the number of variations in the prion genes of the species or specific polymorphisms? Support your answer. ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ 13. Human diseases are often studied using other animals, particularly mice (Mus musculus). According to your data in Table 1. Comparison of Prion Protein Domain for Select Species, which species has the closest prion protein sequence to humans? Why is the mouse used to study prion diseases instead? ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ Prion Amino Acid Sequences By Protein Domain Lesson 5: Oh…Deer Key to Species 116 Abbreviation Scientific Name Common Name Alc.alc Bos.tau Cer.ela Hom.sap Mus.mus Odo.hem Odo.vir Ovi.ari Pan.tro Alces alces shirasi Bos taurus Cervus elaphus nelsoni Homo sapiens Mus musculus Odocoileus hemionus hemionus Odocoileus virginianus Ovis aries Pan troglodytes North American moose Domestic cow Elk Human House mouse Mule deer White-tailed deer Domestic sheep Chimpanzee Prying Into Prions Prion Amino Acid Sequence Species (1) signal Alc.alc m v k s h i g s w i l v l f v amw s d v g l c Bos.tau m v k s h i g s w i l v l f v amw s d v g l c Cer.ela m v k s h i g s w i l v l f v amw s d v g l c Hom.sap m a n l - - g c wm l v l f v a t w s d l g l c Mus.mus m a n l - - g y w l l a l f v t mw t d v g l c Odo.hem m v k s h i g s w i l v l f v amw s d v g l c Odo.vir m v k s h i g s w i l v l f v amw s d v g l c Ovi.ari m v k s h i g s w i l v l f v amw s d v g l c Pan.tro m a n l - - g c wm l v l f v a t w s d l g l c Species (23) pre repeat Alc.alc k k r p k p g g gwn t g g s r y p g q g s p g g n r y p Bos.tau k k r p k p g g gwn t g g s r y p g q g s p g g n r y p Cer.ela k k r p k p g g gwn t g g s r y p g q g s p g g n r y p Hom.sap k k r p k p g g - wn t g g s r y p g q g s p g g n r y p Mus.mus k k r p k p g g - wn t g g s r y p g q g s p g g n r y p Odo.hem k k r p k p g g gwn t g g s r y p g q g s p g g n r y p Odo.vir k k r p k p g g gwn t g g s r y p g q g s p g g n r y p Ovi.ari k - r p k p g g gwn t g g s r y p g q g s p g g n r y p Pan.tro k k r p k p g g - wn t g g s r y p g q g s p g g n r y p Species (51) octapeptide repeat Alc.alc p q g g g g w g q . p h g g g w g q . p h g g g w g q . p h g g g wg q . p h g g g g w g q Bos.tau p q g g g g w g q . p h g g g w g q . p h g g g w g q . p h g g g wg q . p h g g g g w g q Cer.ela p q g g g g w g q . p h g g g w g q . p h g g g w g q . p h g g g wg q . p h g g g g w g q Hom.sap p q g g g g w g q . p h g g g w g q . p h g g g w g q . p h g g g wg q . p h g g g - w g q Mus.mus p q g g - t w g q . p h g g g w g q . p h g g s w g q . p h g g s wg q . p h g g g - w g q Odo.hem p q g g g g w g q . p h g g g w g q . p h g g g w g q . p h g g g wg q . p h g g g g w g q Odo.vir p q g g g g w g q . p h g g g w g q . p h g g g w g q . p h g g g wg q . p h g g g g w g q Ovi.ari p q g g g g w g q . p h g g g w g q . p h g g g w g q . p h g g g wg q . p h g g g g w g q Pan.tro p q g g g g w g q . p h g g g w g q . p h g g g w g q . p h g g g wg q . p h g g g - w g q Mule Deer Lesson 5: Oh…Deer P r y i n g I n t o P r i o n s 117 Lesson 5: Oh…Deer 118 Prying Into Prions Prion Amino Acid Sequence Elk Species (92) Core Alc.alc g g - t h s qw n k p s k p k t nmk h v a g a a a a g a v v g g l g g Bos.tau g g - t h s qw n k p s k p k t nmk h v a g a a a a g a v v g g l g g Cer.ela g g - t h s qw n k p s k p k t nmk h v a g a a a a g a v v g g l g g Hom.sap g g g t h s qw n k p s k p k t nm k hma g a a a a g a v v g g l g g Mus.mus g g g t h n qw n k p s k p k t nm k hma g a a a a g a v v g g l g g Odo.hem g g - t h s qw n k p s k p k t nmk h v a g a a a a g a v v g g l g g Odo.vir g g - t h s qw n k p s k p k t nmk h v a g a a a a g a v v g g l g g Ovi.ari g g - s h s qw n k p s k p k t nmk h v a g a a a a g a v v g g l g g Pan.tro g g g t h s qw n k p s k p k t nm k hma g a a a a g a v v g g l g g Species (128) beta 1 Species (132) loop 1 Alc.alc YML G Alc.alc s ams r p l i h f g n Bos.tau YML G Bos.tau s ams r p l i h f g s Cer.ela YML G Cer.ela s ams r p l i h f g n Hom.sap YML G Hom.sap s ams r p i i h f g s Mus.mus YML G Mus.mus s a m s r p mmh f g n Odo.hem YML G Odo.hem s ams r p l i h f g n Odo.vir YML G Odo.vir s ams r p l i h f g n Ovi.ari YML G Ovi.ari s ams r p l i h f g n Pan.tro YML G Pan.tro s ams r p i i h f g s Species (144) alpha 1 Species (155) loop 2 Alc.alc D Y E D R Y Y R E NM Alc.alc y r y pnq Bos.tau D Y E D R Y Y R E NM Bos.tau h r y pnq Cer.ela D Y E D R Y Y R E NM Cer.ela y r y pnq Hom.sap D Y E D R Y Y R E NM Hom.sap h r y pnq Mus.mus DW E D R Y Y R E NM Mus.mus n r y pnq Odo.hem D Y E D R Y Y R E NM Odo.hem y r y pnq Odo.vir D Y E D R Y Y R E NM Odo.vir y r y pnq Ovi.ari D Y E D R Y Y R E NM Ovi.ari y r y pnq Pan.tro D Y E D R Y Y R E NM Pan.tro h r y pnq Lesson 5: Oh…Deer P r y i n g I n t o P r i o n s 119 Lesson 5: Oh…Deer 120 Prying Into Prions Prion Amino Acid Sequence Species (161) beta 2 Species (165) loop 3 Alc.alc VYYR Alc.alc pv d qynnqn t f v hd Bos.tau VYYR Bos.tau pv d qy snqnn f v hd Cer.ela VYYR Cer.ela pv d qynnqn t f v hd Hom.sap VYYR Hom.sap pmd q y s n q n n f v h d Mus.mus VYYR Mus.mus pv d qynnqnn f v hd Odo.hem VYYR Odo.hem pv d qynnqn t f v hd Odo.vir VYYR Odo.vir pv d qynnqn t f v hd Ovi.ari VYYR Ovi.ari pv d qy snqnn f v hd Pan.tro VYYR Pan.tro pmd q y s s q n n f v h d Species (179) alpha 2 Species (194) Asn Alc.alc C V N I T V KQH T VT T T T Alc.alc kgenf t Bos.tau CVN I TVKEHTVT TT T Bos.tau kgenf t Cer.ela C V N I T V KQH T VT T T T Cer.ela kgenf t Hom.sap C V N I T I KQH T VT T T T Hom.sap kgenf t Mus.mus C V N I T I KQH T VV T T T Mus.mus kgenf t Odo.hem C V N I T V KQH T VT T T T Odo.hem kgenf t Odo.vir C V N I T V KQH T VT T T T Odo.vir kgenf t Ovi.ari C V N I T V KQH T VT T T T Ovi.ari kgenf t Pan.tro C V N I T I KQH T VT T T T Pan.tro kgenf t Species (200) alpha 3 Species (218) pre GPI Alc.alc E T D I K - MME R V V E Q MC I T Q Alc.alc yq r esqayyq- r ga s- - v i l f Bos.tau E T D I K - MME R V V E Q MC I T Q Bos.tau yq r esqayyq- r ga s- - v i l f Cer.ela E T D I K - MME R V V E Q MC I T Q Cer.ela yq r eseayyq- r ga s- - v i l f Hom.sap E T D V K - MME R V V E Q MC I T Q Hom.sap y e r e s q a y y q - r g s sm - v - l f Mus.mus E T D V K - MME R V V E Q MC V T Q Mus.mus yqkesqayydgr r s ss t v - l f Odo.hem E T D I K - MME R V V E Q MC I T Q Odo.hem yq r esqayyq- r ga s- - v i l f Odo.vir E T D I K - MME R V V E Q MC I T Q Odo.vir yq r esqayyq- r ga s- - v i l f Ovi.ari E T D I K - I M E R V V E Q MC I T Q Ovi.ari yq r esqayyq- r ga s- - v i l f Pan.tro E T D V K - MME R V V E Q MC I T Q Pan.tro y e r e s q a y y q - r g s sm - v - l f Moose Lesson 5: Oh…Deer P r y i n g I n t o P r i o n s 121 Lesson 5: Oh…Deer 122 Prying Into Prions Notes ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ Lesson 5: Oh…Deer ________________________________________ P r y i n g I n t o P r i o n s 123 Lesson 6 Educator’s Overview Here To Stay, Not Gone Tomorrow Summary Duration One or two 45-minute class periods Vocabulary The first step in controlling and possibly eradicating CWD is to learn as much as possible about the transmission of the disease. Students read about three experiments that examine the transmission of CWD in deer, elk, and moose. They then consider how results from those studies inform how the Colorado Division of Wildlife manages the disease to minimize its impact on wild, free-ranging deer, elk, and moose populations and protect the public welfare. Learning Objectives Autopsy Cervids Control group Controlled experiment Dependent variable Epidemiologic studies Epidemiologist Euthanize Excreta Experiment Immunology L e s s o n 6 : H e r e To S t a y, N o t G o n e To m o r r o w Independent variable 124 Pharmacology Wildlife ecology After completing this activity, students will be able to: • Identify the control group, independent variable, and dependent variable in three chronic wasting disease research studies • Describe three or more actions that wildlife managers have taken to limit the spread of chronic wasting disease • Describe simple steps that a hunter can take to limit his or her exposure to prions from CWDinfected animals • Describe three or more CWDrelated research studies that Colorado Division of Wildlife staff are involved in and the importance of these studies to managing CWD Background Since the appearance of bovine spongiform encephalopathy (BSE) in the mid-1980s and, especially since the 1996 announcement of an Prying Into Prions apparent relationship between BSE and variant Creutzfeldt-Jakob disease (vCJD), there has been considerable media, public, and animal and human health agency interest in transmissible spongiform encephalopathies like chronic wasting disease. Recent research demonstrates that CWD will pose significant challenges for wildlife managers attempting to control or eradicate the disease. These challenges are the focus of this activity. The three studies included in this activity show that 1.) CWD prions remain in the environment for a long time after infected deer, elk, and moose are gone, 2.) Prions from infected animals are transmissible to healthy animals through saliva and blood, 3.) No tissue from CWD-infected cervids can be considered free of diseasecausing prions, and 4.) No one can predict with absolute certainty that CWD will never cause human disease. Wildlife managers cannot completely control the spread of CWD and cannot entirely eradicate the disease. However, CWD can be managed. The second part of this activity details how the Colorado the development of new diagnostic tests and possibly treatments, and continue to provide the basis for sound decision-making processes that protect animal and human health. Teaching Strategies 1. Thoroughly read the student materials for Here To Stay, Not Gone Tomorrow. Visit the Colorado Division of Wildlife web site to become familiar with the information and materials related to CWD on the site. 2. Decide whether you will be photocopying information from the current Colorado Big Game Regulations, printing information from the DOW web site, or giving students access to the internet to complete this activity. Prepare for the lesson accordingly. 3. Write these three vocabulary words in a visible place: control group, dependent variable, and independent variable. 4. Ask students what the three words pertain to and what they mean. Most students will recognize that these words have something to do with experimental design and the way scientists investigate questions and gain knowledge. Most scientific processes include collecting observations, asking questions about those observations, making predictions, designing experiments to test those predictions, and drawing some sort of conclusion from the results. The results often lead to still other questions. 5. Discuss the idea of a controlled experiment—a planned procedure to test a hypothesis or prediction. A control group is a group in an experiment that receives no experimental treatment. The control and experimental groups are designed to be identical Materials and Preparation • Student Pages: Here To Stay, Not Gone Tomorrow— one photocopy per student • DVD: Managing Chronic Wasting Disease • Access to the Internet • Access to a DVD player and monitor • Current Colorado Big Game Regulations— one copy per student of the 1–2 pages of information concerning Chronic Wasting Disease. A print copy is available at any Colorado Division of Wildlife office or can be downloaded from the DOW Web site at http://wildlife. state.co.us/Rules Regs/Regulations Brochures/ BigGame.htm L e s s o n 6 : H e r e To S t a y, N o t G o n e To m o r r o w Division of Wildlife has attempted to manage the disease to minimize its impact on wild, free-ranging deer, elk, and moose populations and also strives to help protect public welfare. The spread of CWD can largely be prevented through several strategies used by the Colorado Division of Wildlife. Managers selectively cull diseased animals to reduce the overall density of cervids (deer, elk, and moose) in the infected area and to slow the transmission of the disease. The agency also has an extensive surveillance program to examine hunter-harvested deer, elk, and moose and identify new areas of concern. Colorado has implemented regulations that only allow boned meat, quarters (without spinal column or head) or processed meat from hunterharvested deer, elk or moose to be transported out of certain CWD areas. To help protect the public, the DOW works with public health authorities to provide scientifically accurate information regarding chronic wasting disease, as well as simple strategies to lower the risk of exposure to the disease. This information is continually updated and available on the DOW’s web site and in print materials. The agency informs all media outlets— print, radio, and television—of new discoveries or breakthroughs in management. Hunters are able to have their harvested animals tested for CWD, insuring that they can make informed decisions about consuming the meat. Most importantly, Division of Wildlife researchers and biologists are studying chronic wasting disease on numerous fronts— addressing wildlife health issues and assisting public health experts and scientists with their ongoing research. It is anticipated that new research will provide greater insight into CWD and other TSEs, lead to P r y i n g I n t o P r i o n s 125 except for one factor or variable. The factor that is changed or is different in an experiment is called the independent variable. The result, or the variable that is measured, is called the dependent variable. 6. Tell students that the reading for this lesson will contain information from three experiments Control Group related to chronic wasting disease. In addition to the questions that they will be asked in the activity, tell students that they should draw a chart like the one below on a sheet of paper, and identify the control group and the independent and dependent variables for each experiment. Independent Variable Dependent Variable First Experiment Second Experiment Third Experiment 7. Allow students to read the article silently on their own, fill in the chart, and answer the questions. L e s s o n 6 : H e r e To S t a y, N o t G o n e To m o r r o w 8. If time allows, discuss student answers in class. 126 9. Tell students that they will now see Colorado Division of Wildlife researchers and wildlife managers talking about their work and demonstrating how they test for CWD. Inform students that many scenes on the DVD are graphic—they will see tissues being removed from animal heads and lab work as it is performed by DOW personnel. Most students will not feel uncomfortable about the presentation if they are not surprised by what Prying Into Prions they will see. Show the DVD: Managing Chronic Wasting Disease and then discuss as a class. Extensions 10. Each year, around 10,000 deer, elk, and moose heads are submitted for CWD testing. When the program first started, more than double this amount of heads were submitted. As more data became available, researchers and hunters alike recognized that CWD incidence was very low to non-existent in most parts of the state. However, there are a few hot spots. If there is time, have students go the DOW Web site and identify areas of the state where there is a high prevalence of CWD. • Division of Wildlife staff serves nationally as a source of information and resources concerning chronic wasting disease research. Scientific publications of current CWD research are available at our web site at http://wildlife.state.co.us/Hunting/ BigGame/CWD/CWDResearchArticles.htm Depending on the level of your students, you may want to choose several of those studies to review in depth. Students can discuss experimental design, assumptions made, questions raised by the results of the research, and so on. Assessment Students should be able identify the control group, independent, and dependent variables for each of the three experiments described in the text. Key Independent Variable Dependent Variable First Experiment Pasture that had never had CWD infected animals Pasture with decomposing carcass from a deer that had died from CWD placed in it, one with a live CWDinfected deer in it, and one that had been previously occupied by CWDinfected deer Whether or not healthy animals placed in the pastures got CWD Second Experiment Deer exposed to a.) saliva, b.) feces and urine, c.) blood, or d.) brain tissue from healthy wild or captive deer Deer exposed to a.) saliva, b.) feces and urine, c.) blood, or d.) brain tissue from wild or captive deer with CWD Whether or not the exposed deer got CWD Third Experiment Researchers injected the extract of leg muscle from healthy deer into the brains of geneticallyaltered mice Researchers injected the extract of leg muscle from CWDinfected deer into the brains of genetically-altered mice Whether or not the mice got a transmissible spongiform encephalopathy L e s s o n 6 : H e r e To S t a y, N o t G o n e To m o r r o w Control Group P r y i n g I n t o P r i o n s 127 1. Why would these researchers have a type “D” pasture in their experiment? Every experiment needs a control group, one that doesn’t receive any experimental treatment. Each experimental treatment is an independent variable, and the result of each treatment—getting CWD or not—is the dependent variable. 7. Both the web site and the Big Game Regulations provide maps and information that lets hunters know where CWD-infected animals have been found. Why is this important? Hunters will know to take extra precautions when handling the carcass, or, they may choose to hunt in a different location in the state. 2. Why did researchers inoculate the fifth set of tame deer with a.) saliva, b.) feces and urine, c.) blood, or d.) brain tissue from healthy wild deer? The fifth set of deer was the control group. 8. Simple precautions are advised when handling deer, elk, and moose killed in units where the disease has been detected. What are these simple precautions? 3. The urine- and feces-exposed deer did not get sick. Does that mean that deer, elk, and moose cannot get CWD from exposure to urine and feces from infected animals? No, it does not mean that. The sample size (number of deer tested) may have been too small or the study might have been too short for the disease to emerge, or the genetic variety of the fawns might have influenced the results. L e s s o n 6 : H e r e To S t a y, N o t G o n e To m o r r o w 4. Why would researchers be so interested in testing muscle tissue for CWD prions? Humans contract variant Creutzfeldt Jakob disease (vCJD) by eating meat products, which are muscle tissue, from cattle with bovine spongiform encephalopathy (BSE). 128 5. To test the infectivity of skeletal muscle, the researchers used genetically-altered mice that had normal cervid prion protein (CerPrP), which made them susceptible to CWD. Researchers injected the extract of leg muscle from CWD-infected deer into the brains of one group of these special mice. They injected a second group with leg muscle extract from CWD-free mule deer. Why did the researchers have two groups of mice? The second group was the control group. 6. Regardless of where the animal is harvested, what portions of deer, elk, and moose should NOT be consumed? Research indicates that prions accumulate in certain parts of infected animals at early stages in the disease—the brain, eyes, spinal cord, lymph nodes, tonsils, pancreas and spleen. Those portions of the animal should not be consumed. Prying Into Prions • Do not shoot, handle or consume any animal that appears sick. • Wear rubber gloves when field dressing and processing animals. • Bone out the meat from your animal. Minimize the handling of brain and spinal tissues. • Wash hands and instruments thoroughly after field dressing is completed. • Avoid consuming brain, spinal cord, eyes, spleen, tonsils, pancreas and lymph nodes of harvested animals. Normal field dressing, coupled with boning out a carcass, will remove most, if not all, of these body parts. Cutting away all fatty tissue will remove remaining lymph nodes. • Do not consume meat or organs from animals known to be infected with CWD. • Disinfect knives, saws and cutting table surfaces by soaking in a solution of 50 percent unscented household bleach and 50 percent water for an hour. Afterward, allow them to air dry. 9. What portion of the animal is submitted for testing? The animal’s head, which should be removed two to four inches below the point where the neck joins the skull (below the first vertebrae), should be submitted for testing. Lymph nodes in the upper throat are used for testing. The brain stem (not brain) can also be used, so it is important to submit the entire head for testing. Both lymph nodes and brain stem are used for moose testing. Whole brains or pieces of brain tissue cannot be used for testing. 10. Why is it mandatory to submit moose tissue for CWD testing? CWD testing is mandatory for moose to provide more data on CWD in this species. • Ongoing studies to determine if chronic wasting disease can be passed to bighorn sheep, mountain lions and other animals. 11. What happens when an animal tests positive for CWD? Hunters whose deer or elk test positive are eligible for either a license fee refund, an antlerless license for the same species in a remaining season, or an antlerless license for the original unit and species during a season next year. Moose hunters whose animals test positive can get a license fee refund or a cow moose license for the same unit. • Specific studies to determine the ability of chronic wasting disease to infect cattle. 12. Looking again at the DOW web page, what are some of the research projects that DOW employees are involved in? Employees have been involved in the following chronic wasting disease work: • The successful development of improved, more sensitive testing procedures to detect chronic wasting disease in deer, elk and moose. • An ongoing field study designed to measure the relationship between deer density and disease prevalence. • Ongoing research to track the progression of the disease through a deer’s body to better understand how the disease is transmitted and how it can be better diagnosed. • Epidemiological studies conducted by state and federal agencies to determine if a link between chronic wasting disease and human neurological disorders exists. • Laboratory studies to assess the potential susceptibility of different animal species, including humans, to chronic wasting disease. • Ongoing monitoring studies to determine geographic distribution and level of prevalence of chronic wasting disease in the state. • Research into early detection methods to diagnose chronic wasting disease in live, healthy-appearing animals. • Studies of deer movement patterns to determine if links between disease prevalence and deer movement exist. L e s s o n 6 : H e r e To S t a y, N o t G o n e To m o r r o w P r y i n g I n t o P r i o n s 129 Lesson 6 Student Pages Here To Stay, Not Gone Tomorrow Deer, elk, or moose with chronic wasting disease have been found in 14 US states and two Canadian provinces. It is clear that CWD spreads from animal to animal and place to place. But how? L e s s o n 6 : H e r e To S t a y, N o t G o n e To m o r r o w Since CWD was first noticed in captive animals at Colorado’s Foothills Wildlife Research Facility and Wyoming’s Sybille Research Unit, it was reasonable to guess that the disease could only be transmitted between animals that were in close contact with each other. Both facilities tried hard to eradicate CWD. The Sybille center killed all the deer and elk in the affected area. They waited a year to bring new, disease-free animals into the area. Four years later, these healthy deer and elk started showing signs of CWD. 130 The Fort Collins Wildlife Research Facility took more drastic action. First, the affected deer and elk were destroyed. Then, researchers turned over several inches of soil and repeatedly sprayed the pastures and fences and other structures with swimming-pool chlorine, which would easily wipe out any bacteria or viruses. After waiting a year, they brought in 12 healthy elk calves. Three years later, one of these elk contracted CWD. Permanent Environmental Contamination? The idea that the infectious agent for CWD could permanently contaminate both research facilities was disturbing, to say the least. Historically, wildlife managers Prying Into Prions have controlled the spread of wildlife diseases one of three ways. • If the disease is treatable, biologists can either put out medicated feed to treat herds, or trap and treat individual sick animals. For example, wildlife managers will often put out fenbendazolemedicated bait in winter to control lungworm disease in Rocky Mountain bighorn sheep. • If a vaccine is available to prevent the disease, biologists can inoculate vulnerable animals. • If the first two options are not possible, managers can cull ill animals to prevent the spread of the disease. It was apparent that controlling and possibly eradicating CWD would be much more difficult if the pathogen—the prions—stayed in the environment years after the diseased animals were gone. Identifying Prion Sources— The First Experiment How did the prions get on the soil or pasture? No one knew. Colorado Division of Wildlife veterinarians Michael Miller and Lisa Wolfe, and Elizabeth Williams and Thompson Hobbs of CSU set up an experiment to find out. They placed healthy deer in one of four types of pasture: A. One type of pasture had not previously had deer or elk in it. One live CWD-infected deer was put in the pasture. B. One type of pasture had not had live CWD-infected deer in it, but had a decomposed carcass from a deer that had died from CWD placed in it. C. One type of pasture had been previously occupied by CWDinfected deer (more than two years earlier) and had what remained of the deer’s excreta (urine, feces, and saliva). D. One type of pasture had never been occupied by CWD-infected deer. 1. Why would these researchers have a type “D” pasture in their experiment? ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ Identifying Prion Sources— The Second Experiment More research was needed to determine what deer excreta contained the prions that contaminated pasture type C. Researchers from the Colorado Division of Wildlife and Colorado State University set up a second experiment. They tested four groups of 6-month-old hand-raised deer, exposing them to a) saliva, b) feces and urine, c) blood, or d) brain tissue from wild or captive deer with CWD. A fifth set were exposed to each of those materials from wild deer without CWD. The fawns were not genetically identical. 2. Why did researchers inoculate the fifth set of tame deer with a.) saliva, b.) feces and urine, c.) blood, or d.) brain tissue from healthy wild deer? ______________________________________ The veterinarians observed the deer closely. One observation was that the deer in pasture type B did not eat any of the decomposed carcass remains, but did eat the vegetation around it. Deer in all of the other types of pasture fed and interacted normally. After one year, they tested the deer for CWD. All of the deer in pasture type D were still healthy and CWD-free. Two of the ten deer in pasture type A had contracted CWD. Three of the 12 deer in pasture type B had the disease and one of the nine deer in pasture type C was infected with CWD. The presence of CWD prions in saliva may explain why the disease spreads so easily. Deer are social animals that nuzzle, lick and groom one another. But direct deer-to-deer contact is apparently not necessary for transmission. Healthy deer entering areas that once held sick deer can contract CWD because the saliva from the diseased deer remains on the grass and other surfaces. It was not surprising to researchers that healthy deer given a single large transfusion of blood from CWD-infected L e s s o n 6 : H e r e To S t a y, N o t G o n e To m o r r o w All of the conditions that were mimicked in pasture types A, B, and C exist in the wild. Deer will often encounter the carcasses and excreta of other deer. Also, deer usually group together in the winter, so transmission between infected deer and healthy deer is likely. It seemed that CWD would be a tough disease to manage. The deer were observed for 18 months and their tonsil tissue was regularly tested for signs of infection. After 18 months the deer were euthanized (killed humanely), and their brains were removed and examined after death (autopsied). All the deer exposed to saliva and to blood from CWD-infected deer were infected, while those exposed to urine and feces remained healthy. P r y i n g I n t o P r i o n s 131 deer would get the disease. Studies showed that scrapie could be transmitted between sheep that way. From human studies, it was already known that people who received blood or other tissues from victims of variant Creutzfeldt-Jakob disease (vCJD) contracted the illness. But, the findings suggest that it is possible that CWD could be spread to healthy deer, elk, or moose by blood-sucking insects like mosquitoes or ticks. There will need to be more research to find out for sure. 3. The urine- and feces-exposed deer did not get sick. Does that mean that deer, elk, and moose cannot get CWD from exposure to urine and feces from infected animals? ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ L e s s o n 6 : H e r e To S t a y, N o t G o n e To m o r r o w Identifying Prion Sources— The Third Experiment 132 From the beginning, researchers were aware that CWD prions concentrate in certain tissues, such as the brain, spinal cord, lymph nodes, and spleen. They were not sure that CWD prions were present in other tissues or if they were present in concentrations too low to detect. As CWD-infected cervids (the family of animals that includes deer, elk, and moose) were found in an increasingly wide geographic area, and concern about potential zoonotic transmission of CWD increased, public health officials were especially interested in testing muscle tissue for prions. Prying Into Prions 4. Why would researchers be so interested in testing muscle tissue for CWD prions? ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ 5. To test the infectivity of skeletal muscle, the researchers used geneticallyaltered mice that had normal cervid prion protein (CerPrP), which made them susceptible to CWD. Researchers injected the extract of leg muscle from CWDinfected deer into the brains of one group of these special mice. They injected a second group with leg muscle extract from CWD-free mule deer. Why did the researchers have two groups of mice? ______________________________________ The researchers found that the mice injected with muscle samples from infected deer showed the progressive mental dysfunction associated with transmissible spongiform encephalopathies (TSEs) within 18 months. The skeletal muscle from uninfected deer didn’t cause disease when injected into the special mice. A Public Health Problem? The presence of infectious CWD prions in the muscle tissue did raise some issues. Were prions present in meat in enough quantity to be of public health significance? Would people who consumed CWD-contaminated meat from infected deer get some type of TSE? So far, this seems unlikely. Epidemiologic studies, studies of the factors determining and influencing the frequency and distribution of disease, injury, and disability in a population, suggest that the risk of transmission of CWD to humans is very low. Nevertheless, there is no way to prove that a human prion disease associated with CWD cannot appear in the future. Relatively few people in the United States and Canada eat meat from deer, elk, or moose. Most can now test the animals they have harvested for CWD (you’ll find out about this). Since few people have been exposed to CWDcontaminated meat, and since individual genetics influence susceptibility to prion diseases, there may never be enough vulnerable people exposed to CWD to result in a clinically recognizable human disease. Since prion diseases have a long incubation period, epidemiologists (people who do epidemiologic studies) will need to continue surveillance for human prion diseases, particularly in areas where CWD has been detected. The Big Picture All of this research has led to several conclusions: • CWD prions may remain in the environment for a long time after infected deer, elk, and moose have left the area. • No tissue from CWD-infected cervids can be considered free of diseasecausing prions. • No one can predict with absolute certainty that CWD will never cause human disease, although all research to date suggests this is impossible. • Preventing the spread of chronic wasting disease beyond historically infected areas. • Reducing chronic wasting disease prevalence within infected areas by removing deer and elk from diseased herds. • Enforcing illegal feeding regulations. It is illegal to feed wildlife. When people feed deer or elk, they do not help the animals. Instead, they increase the chances that unhealthy animals will spread disease. • Enforcing transport laws that prevent people from moving deer, elk and moose from infected areas or from other states into disease-free areas. In Colorado, people often raise herds of these animals to supply restaurants and specialty grocers with meat. In the past, transporting infected animals from one game ranch to another has been a problem. • Continuing research with other agencies and states to further knowledge about CWD and to improve management of affected deer, elk and moose herds. To inform the public, the DOW works with public health authorities to provide current CWD information and recommendations on its web site, in its print publications, and to newspaper, radio and television agencies. L e s s o n 6 : H e r e To S t a y, N o t G o n e To m o r r o w • Wildlife managers probably cannot completely control the spread of CWD and cannot entirely eradicate the disease. Even though chronic wasting disease is serious and cannot be completely eliminated, state wildlife agencies like the Colorado Division of Wildlife (DOW) may still attempt to manage the disease and protect herds of deer, elk, and moose. The Division of Wildlife’s disease management efforts are focused on: P r y i n g I n t o P r i o n s 133 Educating Hunters and Others Who Eat Deer, Elk, and Moose Meat 8. Simple precautions are advised when handling deer, elk, and moose killed in units where the disease has been detected. What are these simple precautions? While studies do not show any relationship between CWD and human health problems, the DOW takes extra steps to ensure that people who eat deer, elk, and moose meat are informed. You will now look at both the DOW web site and at some pages in the Big Game Regulations that advise hunters and others. Look at the Big Game Regulations and/or this DOW web page to answer the following questions: ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ http://wildlife.state.co.us/ Hunting/BigGame/CWD/ 6. Regardless of where the animal is harvested, what portions of deer, elk, and moose should probably NOT be consumed? ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ L e s s o n 6 : H e r e To S t a y, N o t G o n e To m o r r o w 7. Both the web site and the Big Game Regulations provide maps and information that lets hunters know where CWDinfected animals have been found. Why is this important? 134 ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ Prying Into Prions 9. What portion of the animal is submitted for testing? ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ 10. Why is it mandatory to submit moose tissue for CWD testing? ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ 11. What happens when an animal tests positive for CWD? ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ The Future of CWD Research and Management in Colorado While chronic wasting disease is not going to go away anytime soon, scientists are learning more about CWD and other prion diseases every day. Amazing research in the areas of protein folding, genetics, immunology (study of the immune system), pharmacology (the use of drugs to prevent and treat disease) and wildlife ecology (the study of the interaction of wildlife and its environment) may someday allow wildlife managers to control this disease. Division of Wildlife researchers and biologists are studying chronic wasting disease on numerous fronts—addressing wildlife health issues and assisting public health experts and scientists with their ongoing research. 12. Looking again at the DOW web page, what are some of the research projects that DOW employees are involved in? ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ L e s s o n 6 : H e r e To S t a y, N o t G o n e To m o r r o w The work of some DOW researchers is internationally renowned. While some staff researchers and biologists conduct studies on the disease, others help to collect information from hunters and to keep the public informed. Hundreds of DOW employees and volunteers work every year to further efforts to gain knowledge about and prevent the spread of CWD. It is the hope of all these hardworking people that you—yes you—will stay informed about this important issue, and that possibly someday you might choose to join them in their work. P r y i n g I n t o P r i o n s 135 Glossary Allele: One of the alternative forms of a gene that governs a characteristic Beta sheet (β-sheet): A sharp-angled, folded secondary protein structure Alpha helix (α-helix): A right-handed coil, one possible secondary protein structure Biopsy: the removal and examination of tissue, cell, or fluids from the living body Amino acid: A molecule made up of a central carbon atom, an amino group, a carboxyl group, a single hydrogen group, and an R group Bovine Spongiform Encephalopathy (BSE): Degenerative and fatal transmissible spongiform encephalopathy disease of the brain in cattle (see TSE) Amino group: The molecule -NH2, an essential component of all amino acids Carboxyl group: The molecule -COOH, an essential component of all amino acids Amino-terminus (N-terminus): The end of a polypeptide chain that is made first and that has a free (unbonded) amino group Carboxyl-terminus (C-terminus): The end of a polypeptide chain that is made last and has a free (unbonded) carboxyl-group Amyloid fibers: PrP res prion molecules stacked up, forming long chains Cervids: The family of mammals which grow and shed antlers yearly that includes deer, elk, and moose Anticodon: Groups of three complementary bases on tRNA that recognize and bind to a codon on the mRNA Assay: A test or procedure to purify a substance and determine what it is made of Codon: Three sequential bases of mRNA that code for a particular amino acid Complementary base pairing: Nucleotide bases that are always paired together (In the DNA double helix, adenine pairs with thymine, guanine pairs with cytosine) Astrocytes: Cells in the brain that digest dead neurons, leaving holes Glossary Autopsy: Removal and examination of tissue, cells, or fluid from a dead organism 136 Prying Into Prions Control group: The group used in an experiment that receives no experimental treatment, and is used as a standard for comparison with the experimental groups Controlled experiment: A planned procedure to test a hypothesis or prediction. An experiment in which the scientist compares the results of a control group to the results of the experimental group(s) Dominant allele: The allele’s traits that are physically expressed in the organism Creutzfeldt-Jakob disease (CJD): Chronic, progressive, fatal disease of the central nervous system of humans, a transmissible spongiform encephalopathy found in humans Epidemiologic studies: Studies of the factors that determine and influence the frequency and distribution of disease, injury, and disability in a population Cross-linkage: (see Disulfide bridge) Strong bonds formed by cysteine R groups linked together Dehydration synthesis: A bond that forms between two molecules by the removal of water (one oxygen and two hydrogen molecules). Also known as a dehydration reaction, dehydration linkage, or condensation reaction Emerging disease: A new disease Epidemiologist: A person who conducts epidemiologic studies Eukaryotes: Complex cells that contain a nucleus Euthanize: To kill humanely Excreta: Excretions from the body of an organism such as urine, feces, and saliva Deoxyribonucleic acid (DNA): A polymer made up of four similar chemicals called nucleotides: adenine, thymine, guanine, and cytosine Experiment: A methodic test used to deduce information about the surrounding environment Dependent variable: The measurable result of the experiment. It changes with alteration of the independent variable Familial disease: A disease which is passed down genetically from parent to offspring Dipeptide: Two molecules linked by a peptide bond Fold: The signature shape of a protein that defines its function Disulfide bridges or cross-linkages: Strong bonds formed by cysteine R groups linked together Gene: A unit of genetic information that provides the instructions for a single inherited property or characteristic of an organism—a protein Glossary Domain: Short sections of the polypeptide chain that fold into recognizable shapes P r y i n g I n t o P r i o n s 137 Genetic code: The set of rules by which information encoded in hereditary material in each cell of an organism makes all of the proteins needed by the organism Genome: The entire DNA of an organism Infect: The action of a disease-causing pathogen entering the body Heritable: Changes in DNA that can be passed on from parent to offspring Loop (or Turn): One possible secondary structure in a polypeptide chain (protein) Heterozygous: Having two different forms of an allele Messenger RNA (mRNA): The RNA molecule that carries genetic information from the genes (DNA) to the ribosome Homozygous: Having two of the same forms of an allele Human Genome Project: A worldwide project started in 1990 with the mission of compiling the entire genetic sequence of humans and other organisms Hydrogen bond: Weak attractions between the positively-charged hydrogen of the amino group of one amino acid and the negatively-charged oxygen of a carboxyl group of another Hydrogen group: A single hydrogen atom attached to the center carbon molecule of an amino acid Hydrophobic: Portions of a molecule that are not charged and repel water Glossary Immunology: The study of the immune system 138 Independent variable: The factor in the experiment that is changed by the scientists Prying Into Prions Mutation: A change in the genetic information Nontranscribed strand (Noncoding strand): The strand of a double-stranded DNA that is not used in transcription Nucleotide: Building blocks of DNA or RNA that contain a phosphate group (PO4H), a sugar, and a nitrogen-containing base Pathogen: Disease-producing agent Peptide bond: Type of chemical linkage holding amino acids together, a bond between the carboxyl group of one amino acid and the amino group of another Pharmacology: The study of the use of drugs in preventing and treating disease Polymer: Large molecules made up of smaller molecules Polymorphism: The existence within a population of two or more genetically different forms of a protein Quaternary or fourth-level protein structure: Two or more polypeptides joined together by many different kinds of chemical bonds to make a large, finished protein Polypeptide: A chain of amino acid molecules linked by peptide bonds Primary protein structure: The number, kind, and order of amino acids joined together in a polypeptide chain R group: The molecule that makes the 20 amino acids different from each other. It is attached to the central carbon atom Recessive allele: The allele’s traits that are not physically expressed in the organism Prion: Proteinaceous infectious particle that is responsible for transmissible spongiform encephalopathies such as chronic wasting disease Prion or “protein only” hypothesis: The proposal that a misfolded prion protein could become a pathogenic entity that kills Promoter site: A sequence of DNA bases that identifies the strand to be transcribed, the site where the RNA polymerase enzyme attaches Protein: Large compounds made up of one or more polypeptide chains Protein folding problem: The struggle of trying to uncover the mechanism that directs protein folding Protein synthesis: The making of protein in the cell through the process of DNA transcription Ribosome: Organelle in the cell responsible for protein synthesis Ribonucleic acid (RNA): A polymer of nucleic acids similar to DNA but containing the base uracil instead of thymine, and having a ribose sugar rather than a deoxyribose sugar. It is used in directing protein synthesis outside of the nucleus Ribosomal RNA (rRNA): The central component of the ribosome that helps translate mRNA into amino acids RNA polymerase: The enzyme that unwinds DNA during transcription to make mRNA Secondary protein structure: The shape of an amino acid after hydrogen bonding takes place. The shape can be either a coiling alpha (α) helix or a folded beta (β) sheet, and is determined by the R groups of the amino acids in the chain Glossary Silent (Neutral) mutation: A mutation results in a change in the nucleotide sequence, but the resulting triplet still matches a codon that makes the same amino acid. This type of mutation is harmless P r y i n g I n t o P r i o n s 139 Sporadic disease: Disease that occurs occasionally and at random intervals in a population Tertiary protein structure: A complex folding of protein which determines the resulting function of the protein Transcribed strand (sense strand or coding strand): The strand of double-stranded DNA that is used for transcription Transcription: Process by which genetic information from DNA is converted into its RNA equivalent Transmissible Spongiform Encephalopathies (TSE): Transmissible, fatal diseases of the central nervous system that result in a sponge-like appearance of the brain tissue Triplet: A sequence of three nucleotide bases of DNA that code for a specific amino acid Turn (or Loop): One possible secondary structure in a polypeptide chain (protein) Variant Creutzfeldt-Jakob disease (vCJD): A form of Creutzfeldt-Jakob disease linked to consumption of bovine spongiform encephalopathy (BSE) infected meat products Transfer RNA (tRNA): RNA molecules that carry amino acids to the ribosome Wildlife ecology: The study of the interaction of wildlife and its environment Translation: Making a protein using the information provided by messenger RNA Glossary Zoonotic: A disease that can be transferred to humans from other animals 140 Prying Into Prions